PaxVax CVD 103-HgR single-dose live oral cholera vaccine

ABSTRACT

Introduction: Cholera remains a problem in developing countries and a risk for travelers. Hypochlorhydria, blood group O, cardiac and renal disease increase the risk of developing cholera gravis. Oral vaccines containing inactivated Vibrio cholerae and requiring two doses are available in some countries. No cholera vaccine had been available for U.S. travelers for decades until 2016 when CVD 103-HgR (VAXCHORA™), an oral live attenuated vaccine, was licensed by the U.S. FDA.

Areas covered: Enduring protection following wild-type cholera provided the rationale to develop a single-dose live oral vaccine. CVD 103-HgR is well-tolerated and protects against cholera caused by V. cholerae O1 of either serotype (Inaba, Ogawa) and biotype (El Tor, Classical). Since 90% vaccine efficacy is evident 10 days post-ingestion of a single dose, CVD 103-HgR can rapidly protect travelers. Vibriocidal antibody seroconversion correlates with protection; >90% of U.S. adult (including elderly) vaccinees seroconvert. The U.S. Public Health Service’s Advisory Committee on Immunization Practices recommends CVD 103-HgR for U.S. travelers to areas of ongoing cholera transmission.

Expert commentary: Next steps include evaluations in children, post-licensure safety and effectiveness monitoring, diminishing cold chain constraints, optimizing a ‘high-dose’ formulation for developing countries, and diminishing/eliminating the need for water to administer a dose.

KEYWORDS:

• Cholera
• cholera vaccines
• live oral vaccine
• travelers’ vaccines
• vaccine

1. Introduction

Cholera, a diarrheal disease that in its severe clinical form, cholera gravis, is characterized by the passage of voluminous electrolyte-rich rice-water stools, can lead to dehydration, acute renal failure, and death, even in otherwise healthy adults, if prompt rehydration is not initiated. Serogroup O1 of Vibrio cholerae is currently responsible for >99% of all cholera cases worldwide [1–4] and two main serotypes are isolated, Inaba and Ogawa. There are also two V. cholerae O1 biotypes, classical and El Tor, defined by phenotypic and genotypic characteristics. Over the centuries, cholera has periodically spread in pandemic fashion. Since the 1960s, the world has experienced the progressive dissemination of a seventh pandemic, this one due to El Tor [5]. More recently, ‘El Tor hybrid strains’ have appeared consisting of V. cholerae O1 El Tor expressing classical cholera enterotoxin and sometimes also exhibiting classical biotype toxin co-regulated pili (TCP), the protein organelles on the vibrio surface that foster colonization of the human proximal small intestine [6,7].

Reservoirs of enterotoxigenic V. cholerae O1 include persons with clinical and subclinical infection and certain brackish water environmental niches where the vibrios are attached to resident copepods (zooplankton), crustacea, and other chitinous fauna [8]. Cholera causes explosive epidemics even in endemic areas where cholera is often highly seasonal and erupts in multiple foci simultaneously related to zooplankton blooms. One environmental niche exists in the Gulf of Mexico along the Texas and Louisiana coast where sporadic cases and mini-outbreaks of cholera occur every few years due to a highly hemolytic El Tor strain [9,10].

A recent worldwide burden estimate indicates that 1.3–4.0 million cases of cholera and 21,000–143,000 deaths occur annually [11]. Bacteriologic methods to confirm cases are often unavailable in developing countries. Moreover, only ~5–10% of cholera cases are actually reported because some countries, concerned over the impact on tourism and trade, fail to report cholera in a timely way, if at all.

The incidence of confirmed cholera among travelers from the USA who visit developing countries is generally low but is related to the geographic areas frequented. The number of cases rose markedly when cholera returned to South America in 1991 (160 US cases from 1992 to 1994) [12] and during the 2010 Haiti epidemic (23 cases during the first 6 months of the epidemic [13]. Taylor et al. [14] established systematic bacteriologic culturing for V. cholerae O1 among diarrhea cases attended in the US Embassy’s health clinic in Lima, Peru. Over the 3-year period 1992–1994, the mean annual attack rate for confirmed cholera among US citizens employed at the embassy was 5.3 cases/10³ employees [14]. This amplified bacteriology laboratory-supported passive surveillance identified a cholera risk similar to incidence rates observed in adults in cholera-endemic Bangladesh. Patients ‘presented with a severe form of travelers’ diarrhea,’ although none required intravenous rehydration or hospitalization [14].

Travelers’ cholera can be severe. An outbreak of cholera occurred among 336 passengers on a flight from Lima, Peru to Los Angeles, CA; 194 (58%) passengers were traced in the USA and submitted laboratory specimens [15]. One hundred passengers (52%) had laboratory-confirmed V. cholerae O1 infection (V. cholerae O1 isolation, elevated vibriocidal antibody titer, or both) and 75 of these (75%) reported diarrhea (including 19 US citizens). Ten cases were hospitalized and one case was fatal. Absent the intensive epidemiologic investigation, the fact that an outbreak of severe diarrheal disease had occurred, that it was cholera, and that 10 of the 75 cases of diarrhea (13%) had to be hospitalized in different states and one case was fatal would have been totally missed.

A case of severe cholera was documented in an Italian traveler who acquired disease in Cuba in 2013 and became ill during the return flight to Italy [16]. He developed profuse diarrheal purging, dehydration, and acute renal failure but progressively improved during a 10-day hospitalization that included dialysis.

Persons at increased risk of developing cholera gravis include individuals of blood group O, those with hypochlorhydria (from chronic use of antacids or drugs to diminish gastric acid production or from partial gastrectomy) and persons with chronic cardiac and renal impairment. Also at increased risk are individuals who must travel, work, or reside in places where they are many hours away from reaching health care should they develop severe cholera diarrhea. Purging at 1 liter/hour, even a previously healthy adult is at risk of death within hours unless she/he has access (including rapid transport) to a health facility where rehydration can be initiated.

2. Rationale for a live oral vaccine

In the early 1970s, prevalent dogma taught that an initial clinical episode of V. cholerae O1 infection stimulated minimal, short-lived protection against subsequent exposure and that an efficacious cholera vaccine would have to improve vastly on the protective effect of natural cholera [17]. However, in the mid-1970s, a model of experimental cholera was established in community volunteers (mainly university students) at the Center for Vaccine Development (CVD) of the University of Maryland School of Medicine that allowed intensive studies of the degree of protection elicited by a single bout of cholera when cohorts of volunteers were rechallenged 1–3 months later with the same or with the heterologous serotype within the same biotype [18–21]. In this model, a total diarrheal purge ≥3 liters but <5.0 liters was considered moderate cholera, while a purge ≥5.0 liters was considered ‘cholera gravis.’ Volunteers received oral and/or intravenous rehydration to keep up with ongoing losses of body water and electrolytes. These studies showed that an initial experimental challenge with V. cholerae O1 stimulated powerful protection against rechallenge. This was particularly impressive with classical biotype. Among 41 individuals challenged initially with 10⁶ colony forming units (cfu) of classical Ogawa or Inaba, 22/23 Ogawa and 10/12 Inaba recipients developed cholera (95.6% and 83.3% attack rates, respectively); 4 of 6 individuals challenged with a one-log lower inoculum of classical Ogawa (10⁵ cfu) also developed cholera (67% attack rate) [18]. When 16 classical cholera ‘veterans’ returned for rechallenge 1–3 months after their initial cholera episode, 0/16 developed diarrhea (100% protection). Moreover, 0/16 had V. cholerae O1 detected in direct (without enrichment) coprocultures, indicating a powerful antibacterial effect. Sixteen hours post-challenge, five rechallenge veterans and six controls swallowed intestinal tubes to have proximal jejunal fluid collected for culture every 4 h until diarrhea occurred or until ~24 h had passed. Five of six controls developed diarrhea and each yielded V. cholerae O1 in their jejunal fluid within 8 h before onset of diarrhea (N = 3) or within 1 h after onset (N = 2); these five controls also had positive coprocultures [18]. The sixth control, who failed to develop diarrhea, had negative jejunal fluid and coprocultures. Notably, all jejunal fluid and stool cultures of the protected rechallenged ‘veteran’ subjects failed to grow vibrios. Collectively, these data indicated that the powerful protective effect that follows recovery from an episode of clinical cholera is mediated by potent antibacterial immune mechanisms [18].

Serotype-homologous and serotype-heterologous rechallenge studies with V. cholerae O1 of El Tor biotype similarly showed that an initial episode of clinical cholera conferred high-level (89.5%) protection against subsequent challenge, although the nature of the immunity appeared slightly less potent. Whereas the attack rate for cholera diarrhea was 32/37 (86.5%) among participants challenged for the first time, only 2/22 (9.1%) developed diarrhea upon rechallenge 1–3 months subsequently. Nevertheless, the occasional clinical breakthroughs suggested that El Tor vibrios might be somewhat less immunizing than classical. Support for this hypothesis came from coproculture data. Whereas 0/16 classical rechallenge subjects had positive direct cultures, 8/22 (36.4%) rechallenged El Tor subjects had positive direct coprocultures (p = 0.012 vs. classical rechallenged subjects).

A fundamental question to be answered was the duration of the potent protection elicited by an episode of classical cholera. Four volunteers who experienced an initial cholera episode 33–36 months previously returned for a rechallenge, along with five control subjects [22]. Cholera was observed in 4/5 controls, who purged 2.1, 5.3, 7.1, and 7.2 l and all had positive coprocultures. In contrast, 0/4 rechallenge subjects developed diarrhea (p = 0.04 versus controls) and only 1 had a positive coproculture and shed few V. cholerae O1. This exceptional study documented that the protective effect of an episode of classical cholera endured for at least 3 years and the potent antibacterial immune effect was still evident.

Although component toxin antigens in oral vaccines, such as purified B subunit, that stimulate cholera antitoxin can augment for several months the protection conferred by antigens that stimulate antibacterial immune responses [23], considerable accumulated evidence shows that on balance, antibacterial immunity is the dominant mechanism that mediates protection against cholera [2,21,24,25].

2.1. Serum vibriocidal antibody correlates with protection

Serum vibriocidal antibody has long been recognized as a correlate of protection against cholera. Mosley et al. showed that in East Pakistan (now Bangladesh), cholera incidence was highest in children <5 years of age and then diminished with increasing age [26]. The population geometric mean titer (GMT) of serum vibriocidal antibody rose with age and for each doubling of vibriocidal antibody GMT, there was a halving of cholera incidence [26]. Mosley et al. [27] measured baseline vibriocidal titers in household contacts of cholera patients, followed the contacts prospectively, and found that contacts with higher titers had significantly lower cholera attack rates than low-titer contacts. Subsequent investigators in Bangladesh corroborated Mosley’s pioneer results [28,29].

2.2. V. cholerae O1 serotype epitopes

Since serum vibriocidal antibody following wild-type infection or after immunization with various oral and parenteral cholera vaccines is the strongest correlate of protection, and since vibriocidal antibodies are overwhelmingly directed against V. cholerae O1 LPS antigens, it is appropriate to scrutinize those O antigens. Three B-cell O antigen epitopes, A, B, and C, serologically define the three serotypes of V. cholerae O1 including Inaba (A,C), Ogawa (A,B), and the rare and unstable Hikojima (A,B,C). The structure of V. cholerae O1 O-polysaccharide consists of an α(1 → 2)-linked chain of the amino sugar d-perosamine, that is, 4-amino-4,6-dideoxy-d-mannose, the amino groups of which are acylated with 3-deoxy-l-glycerotetronic acid [30]. A minute difference in the terminal sugar of the O-polysaccharide distinguishes Inaba versus Ogawa serotype. Inaba has a hydroxyl at the second position, while Ogawa has a 2-O-methyl group [31]. The gene wbeT (previously named rfbT) encodes a methyltransferase that methylates the terminal perosamine saccharide resulting in the Ogawa serotype [32]. Certain mutations in wbeT disable methylation of that terminal sugar and thereby convert an Ogawa strain to Inaba serotype. Conversely, specific mutations in Inaba wbeT can convert the strain to Ogawa. The ability of V. cholerae O1 to undergo serotype conversion has been documented in cholera patients and in vitro [32,33]. Serotype changes also occur during epidemics. The enormous 1991 Peruvian cholera epidemic began as El Tor Inaba but later changed to El Tor Ogawa. The small difference in structure between Inaba and Ogawa O polysaccharides, the shared O epitope between the serotypes, the relatively modest difference in vibriocidal antibody titers irrespective of whether Inaba or Ogawa bacteria are the assay targets after Inaba or Ogawa cholera and the solid cross serotype protection observed in volunteer challenge studies collectively offer a theoretical basis for cross serotype protection.

2.3. Evidence of cross serotype protection with early vaccines

How well vaccines of one serotype, that is, Inaba or Ogawa, protect against the other serotype has long been a concern among cholera vaccine developers and implementers. Rechallenge studies in North American volunteers with wild-type virulent V. cholerae O1 within both the classical and El Tor biotypes show that an initial infection with one serotype provides a very high level of protection (90% for studies with El Tor and 100% for classical). While cholera antitoxin may have contributed in part to the protection observed, the majority of evidence indicates that antibacterial immunity predominates in mediating protection and that classical biotype strains elicited somewhat more potent antibacterial mechanisms than El Tor [18,21,24,25]. Epidemiologic field studies in Bangladesh corroborated that an initial classical biotype infection stimulated stronger protection against both classical and El Tor organisms than did initial clinical infection with El Tor [34].

2.3.1. Killed whole cell parenteral vaccines

Two randomized, controlled field trials were performed with serotype-monovalent V. cholerae O1 killed whole-cell parenteral vaccines that provide evidence of cross-serotype protection and its limits [35,36]. In a field trial in rural East Pakistan, 45,771 children < 15 years of age were randomly allocated to 1 of 4 groups to receive 2 doses (1 year apart) of monovalent classical Inaba vaccine, monovalent classical Ogawa vaccine, a purified El Tor Inaba antigen extract that included LPS or control vaccine (tetanus/diphtheria toxoids) [35]. Follow-up surveillance maintained for 30 months showed that virtually all cholera in this locale was due to classical Inaba strains. Since children <5 years of age in this endemic area were at greatest risk of developing cholera, there was particular interest in vaccine performance in that young age group.

Monovalent classical Inaba whole-cell vaccine demonstrated 83.9% vaccine efficacy overall against Inaba cholera during the follow-up, including 96.6% protection among children ≥5 years of age and an impressive 74.6% protection among children <5 years of age. Even during the first year of surveillance, after only one dose of vaccine had been administered, monovalent Inaba whole-cell vaccine conferred 89% protection against Inaba cholera upon children <5 years of age. In contrast, monovalent Ogawa killed whole-cell vaccine failed to protect children <5 years of age against cholera (0% vaccine efficacy). Interestingly, children who were 5–14 years of age at the time of initial vaccination with Ogawa vaccine were significantly protected against Inaba cholera (47.9% VE); even one dose provided measurable cross protection against Inaba (41.0% VE) during the first year of follow-up. It is presumed that the older children who received Ogawa vaccine and exhibited cross serotype protection against Inaba were immunologically primed by prior exposure and their more mature immune responses stimulated by Ogawa vaccine included protective cross reacting antibodies against common O1 antigens.

Another large field trial involving 223,566 participants was carried out in the Philippines where El Tor Ogawa was endemic [36]. By randomly allocating subjects to receive the same killed whole-cell monovalent classical V. cholerae O1 vaccines as had been used in the East Pakistan trial or monovalent killed whole-cell El Tor Inaba or El Tor Ogawa vaccines, the protective capacity of these different types of vaccines could be assessed in preventing El Tor Ogawa cholera. Controls received typhoid vaccine. In this trial, 41.3% of the participants were <10 years of age including 27.0% 5–9 years and 14.3% <5 years of age [36]. During 7 months of follow-up postvaccination, 223 El Tor Ogawa cholera cases were confirmed and all the vaccines conferred a similar level of significant protection. Vaccine efficacies were 71.4% (95% CI, 55.8–81.5) for classical Inaba vaccine; 64.6% (95% CI, 47.2–76.2) for El Tor Ogawa; 60.2% (95% CI, 41.5–73.0) for classical Ogawa; 58.1% (38.7–71.3) for El Tor Inaba. The overall efficacy data from this trial demonstrated unequivocally that classical parenteral whole-cell vaccine can protect against El Tor vibrios and that monovalent Inaba vaccines can protect against heterologous El Tor Ogawa as competently as monovalent Ogawa vaccines. Regrettably, the efficacy data were not presented in relation to age to assess specifically protection among children <5 years of age.

2.4. Vibriocidal antibody immunogenicity guides live vaccine development

Volunteers who participated in early CVD cholera challenge studies mounted strong serum vibriocidal antibody responses that peaked at 10–14 days post-challenge and dropped markedly over the ensuing 4 months [37]. Importantly, the small number of subjects who ingested V. cholerae O1 and shed vibrios but failed to develop diarrhea, that is, had asymptomatic infection also exhibited strong vibriocidal antibody responses [37]. Collectively, the data from the volunteer challenge model indicated that a worthy cholera vaccine development strategy would be to attenuate virulent classical V. cholerae O1 adequately so that the resultant strain does not cause diarrhea and is otherwise well tolerated yet elicits strong serum vibriocidal antibody responses. It was hypothesized that such a strain would make a good live oral vaccine [38].

3. CVD 103-HgR

After clinical trials with a number of candidate deletion mutants of classical and El Tor V. cholerae O1 [25,39], an optimal attenuated strain was engineered [40–42]. Vaccine strain CVD 103-HgR, derived from virulent classical Inaba strain 569 B [18], has a deletion of 94% of ctxA, which encodes the ADP ribosylating A subunit of cholera toxin (CT), while leaving intact ctxB, which encodes the immunogenic nontoxic binding B subunit. At the behest of the WHO, an indelible marker was inserted in the vaccine strain by introducing a gene encoding resistance to Hg++ into hlyA [40,42], which encodes Hemolysin A, considered by some to be a minor pathogenicity factor. The resultant vaccine strain, CVD 103-HgR, expresses classical TCP, exhibits diminished

motility, and induces in cultured human cells significantly less expression of IL-8 and other pro-inflammatory cytokines than reactogenic engineered live oral vaccine strains [43].

3.1. Biosafety relevant to recombinant vaccine strains

As part of the regulatory authority applications for licensure of CVD 103-HgR originally submitted as Orochol Berna, Mutacol Berna, and Orochol E, and more recently as PaxVax’s VAXCHORA, data had to be provided that assessed the hypothetical risks of reacquisition of ctxA which theoretically could revert the vaccine strain to pathogenicity resembling its wild-type parent. The bacterial genetic considerations and extensive experiments that show the very low probability of such reacquisition and the biosafety of the vaccine strain have been reviewed by Viret et al. [44] and by Kaper et al. [41].

Since this vaccine strain is a recombinant genetically modified organism (‘GMO’), bacteriological studies that constitute a quantitative environmental risk assessment also had to be performed to document that the survival of the vaccine strain in various ecological niches including surface water, brackish estuarine water, soil, and various foods was not longer than survival of its wild-type parent. Finally, once field studies with the vaccine were initiated in cholera-endemic countries, since this was considered a deliberate environmental release of a GMO, it was critical to establish not only the degree of fecal shedding by vaccinees but also the ability to recover the vaccine strain from untreated fecally contaminated wastewater [45], its persistence in the environment [45], and the frequency of horizontal transfer of the vaccine strain to household contacts of vaccinees [45]. CVD 103-HgR was found to be shed only minimally by developing country vaccinees [45–48], was not recovered from latrines and sewers adjoining households of vaccinees [45], and was not recovered from vaccinated household contacts [45].

4. Experience with earlier commercial formulations of CVD 103-HgR

The initial CVD 103-HgR commercial formulation was manufactured by the Swiss Serum and Vaccine Institute, Berne (SSVI, BERNA) [40,49,50]. A formulation containing ~5 × 10⁸ cfu per dose of lyophilized vaccine was commercialized under the trade names Orochol® (licensed in Switzerland, Australia, New Zealand, and a number of other countries) and Mutacol® (licensed in Canada). SSVI had already commercialized Ty21a, a widely used live oral typhoid vaccine. Orochol came in a double-sachet presentation with one sachet containing lyophilized vaccine and the other having buffer powder. Contents of the two sachets were mixed in a cup with 100 ml water to make a ‘vaccine cocktail’ that was then ingested.

4.1. Orochol immunogenicity

4.1.1. Serum vibriocidal antibody

Multiple randomized, placebo-controlled trials in North American and European adults showed that Orochol was well tolerated and stimulated seroconversion of vibriocidal antibodies in circa 88–97% of vaccinees with ≥50% reaching a reciprocal titer of ≥2560 and with 70–80% developing significant rises in serum IgG cholera antitoxin [38,40,51–54]. Although ~90% of US and European vaccinees mounted vibriocidal seroconversion, only 17–25% of vaccinees had positive coprocultures [40,51].

4.1.2. Mucosal immune responses to Orochol

Serum vibriocidal antibodies constitute the strongest correlate of protection but they may be a proxy for local intestinal immune responses. Two publications describe the gut-derived trafficking antibody secreting cell (ASC) response to CVD 103-HgR in North American and European subjects who ingested Orochol [55,56]. IgA, IgM, and IgG ASC responses to Inaba LPS in North American adults 7 days after ingesting a dose of Orochol were compared to the responses of unvaccinated controls who had been challenged 7 days earlier with wild-type V. cholerae O1 classical Inaba [55]. Among 26 vaccinees, 15 mounted ASC responses to Inaba LPS and 21 to CT; most responses were IgA. The IgA ASC responses per 10⁶ peripheral blood mononuclear cells (PBMC) were moderate in magnitude (geometric means of 15–39 for LPS and 30–92 for CT). ASC responses of non-immunized control volunteers to Inaba LPS and CT 7 days following challenge with wild-type V. cholerae O1 were similar to those recorded in the vaccinees: 10 of 17 (59%) controls responded to LPS and 10 of 17 (59%) to CT, with geometric mean numbers of ASC of 33–63/10⁶ PBMC for LPS and 34–125/10⁶ PBMC for CT. The differences in responses to live vaccine versus wild-type V. cholerae O1 were not statistically significant. However, significant differences were found in the magnitude of ASC response when vaccinees were challenged with wild-type V. cholerae O1 8 or 30 days after their single dose of CVD 103-HgR. When ASC were measured 7 days after the vaccinees were challenged, their geometric mean numbers of ASC were significantly lower than were seen in the unimmunized controls (p = 0.007 for vaccinees challenged 8 days and p = 0.01 for vaccinees challenged 30 days after vaccination vs. challenged controls). At those time points, the challenged vaccinees exhibited ‘active intestinal immunity’ that blunted the ASC response on repeat exposure to live V. cholerae O1. This ‘active intestinal immunity’ phenomenon has been described with Ty21a live oral typhoid vaccine [57].

Viret et al. [56] reported rates of ASC response to Inaba LPS of 60% for IgA and IgM and 80% for IgG in five Europeans given a single dose of CVD 103-HgR. Their strongest responses were IgM ASC, followed by IgA ASC. When they revaccinated their subjects 14 months later, the ASC responses were starkly muted compared to the initial responses. They also attributed this to active intestinal immunity.

4.2. Protection conferred by Orochol

Eight cohorts of US volunteers vaccinated with a single oral dose of Orochol (N = 103) and 1 cohort vaccinated with 2 doses (10 days apart) were challenged in 7 separate studies with wild-type V. cholerae O1 of Inaba or Ogawa serotype and of classical or El Tor biotype, along with 86 unimmunized controls [40,52,58]. Protection was documented as early as 8 days and as long as 6 months (the longest interval tested) after ingesting a single dose [58]. Since almost all cholera worldwide presently is El Tor biotype or variants, it is important to review how efficacious classical Inaba vaccine strain CVD 103-HgR is in preventing El Tor cholera. Tables 1 and 2 (top rows) summarize composite results of the 4 experimental cholera challenge studies of 5 US cohorts encompassing 64 vaccinees and 47 controls who were challenged with either El Tor Inaba N16961 (1 month or 3 months postvaccination), El Tor Ogawa E7946 (1 month postvaccination), or El Tor Ogawa 3008 (10 days or 1 month postvaccination). Using the primary end point preferred by the US FDA for experimental cholera challenge studies, protection against cholera diarrhea of a severity of ≥3.0 l total purge, there was only 1 breakthrough among 64 challenged Orochol vaccinees; this one vaccinee purged ≥ 3.0 l when challenged with El Tor Inaba N16961 3 months after vaccination [52].

Table 1. Summary of cohorts of Maryland community volunteers immunized with CVD 103-HgR manufactured by the Swiss Serum and Vaccine Institute (Orochol) or by PaxVax (VAXCHORA), followed by challenge of vaccinees and unimmunized control volunteers with virulent Vibrio cholerae O1 of El Tor biotype and Inaba or Ogawa serotype.

Study no. (product)	Year	No. doses of CVD 103-HgR	Colony forming units (cfu) per dose of vaccine	V. cholerae O1 challenge strain	Challenge inoculum (cfu)	Interval between vaccination and challenge	No. of vaccines	No. of controls
CVD 9003 (OROCHOL)	1987	1	~10^8	El Tor Inaba 16961	10^6	1 month	6	8
CVD 9007 (OROCHOL)	1989	2*	~10^8	El Tor Ogawa E7946	10^6	1 month	11	8
CVD 19002 (OROCHOL)	1993	1	~10^8	El Tor Ogawa 3008	10^6	1 month^†	10	8
						10 days^†	9
CVD 45000 (OROCHOL)	1999	1	~10^8	El Tor Inaba N16961	10^5‡	3 months	28	23
NCT01895855 (VAXCHORA)	2013 2014	1	~10^8	El Tor Inaba N16961	10^5‡	10 days	35	66^§
						3 months	33
Totals							132	113

*The two doses of CVD 103-HgR were administered 10 days apart followed by challenge 1 month after the second dose.

^†The Day 10 challenge cohort was vaccinated 20 days before the 1-month challenge cohort and both these cohorts of vaccinees were challenged simultaneously along with a single shared control group.

^‡These challenges utilized a GMP frozen inoculum challenge lot of El Tor Inaba N16961 provided by NIH that enabled simultaneous multisite challenge studies to ensue with the identical inoculum.

^§With agreement of the FDA, the 33 randomized controls challenged at day 10 and the 33 randomized controls challenged at 3 months after immunization were pooled (N = 66 controls) to estimate the efficacy at each time point.

Table 2. Immunization of cohorts of adult Maryland community volunteers with CVD 103-HgR live oral cholera vaccine manufactured by the Swiss Serum and Vaccine Institute (Orochol) or by PaxVax (VAXCHORA), followed by experimental challenge of vaccinees and unimmunized control volunteers with virulent Vibrio cholerae O1 of El Tor biotype and Inaba or Ogawa serotype.

		≥5.0 l	≥3.0 l	≥1.0 l	Any diarrhea
Orochol formulation of CVD 103-HgR*	Vaccinees	1/64 (1.6%)	1/64 (1.6%)	8/64 (12.5%)	19/64 (29.7%)
	Controls	10/47 (21.3%)	34/47 (34.0%)	28/47 (72.3%)	43/47 (91.5%)
	Vaccine efficacy (95% CI)	92.7% (44.6–99.0%)	95.4% (66.6–99.4%)	79.0% (58.2–89.5%)	67.6% (52.2–78.0%)
	p Value^†	0.0019	0.000002	<0.000001	<0.000001
VAXCHORA formulation of CVD 103-HgR, 10-day challenge^‡	Vaccinees	1/35 (2.9%)	2/35 (5.7%)	2/35 (5.7%)	5/35 (14.3%)
	Controls	28/66 (42.4%)	39/66 (59.1%)	54/66 (81.8%)	61/66 (92.4%)
	Vaccine efficacy (95% CI)	93.3% (56.2–100%)	90.3% (61.7–100%)	93.0% (73.1–98.2%)	84.5% (67.0–100%)
	P value^†	0.000078	<0.00001	<0.00001	<0.00001
VAXCHORA formulation of CVD 103-HgR, 3-month challenge^‡	Vaccinees	2/33 (6.1%)	4/33 (12.1%)	6/33 (18.2%)	15/33 (45.5%)
	Controls	28/66 (42.4%)	39/66 (59.1%)	54/66 (81.8%)	61/66 (92.4%)
	Vaccine efficacy (95% CI)	85.7% (46.2–100%)	79.5% (49.1–100%)	77.8% (53.8–89.3%)	50.8% (33.6–66.8%)
	P value^†	0.00014	0.00001	<0.00001	0.000001

*5 Cohorts of US volunteers immunized with a single dose (4 cohorts) or with two doses (1 cohort) of the Orochol formulation of CVD 103-HgR were challenged with El Tor Ogawa or El Tor Inaba in four separate challenge studies at 10 days (El Tor Ogawa 3008), 1 month (El Tor Ogawa E7946, El Tor 3008, or El Tor Inaba N16961), or 3 months (El Tor Inaba N16961) post-immunization (see Table 1).

^†Proportions compared by two-tailed Fisher’s exact test.

^‡Cohorts of healthy adult volunteers at three academic clinical research sites in the USA were challenged with El Tor Inaba N16961 at either 10 days or 3 months post-immunization and compared to the pooled controls from the 10-day and 3-month challenges [59].

Challenge studies were carried out on a Research Isolation Ward under physical containment so all passed stools were collected, examined, and graded on a scale of 1 to 5. Grade 1 is a firm formed stool (fecal bolus), while grade 2 is a soft formed stool. Grade 3 is a thick liquid stool that takes the shape of the container. Grade 4 is an opaque watery stool. Grade 5 is a rice water stool. Grades 3–5 are considered abnormal stools. All stools grade 3 or greater were weighed; 1 g of loose stool was assumed to be equal to 1 ml volume.

Diarrhea as an efficacy end point following challenge was defined as the passage of ≥2 loose stools (grade 3–5) over a 48-h period totaling at least 200 ml, or a single loose stool ≥300 ml.

Moderate or severe cholera diarrhea was defined as the passage of at least 3.0 l or 5.0 l of diarrheal stool, respectively.

4.3. Cross-serotype protection conferred by Orochol and Orochol E

Evidence that classical Inaba CVD 103-HgR protects against cholera due to El Tor Ogawa strains comes from two challenge studies with Orochol and one field study with Orochol E (Section 4.4.2.2) [60]. In 1 volunteer challenge study (Tables 1 and 2), 11 healthy US adults who were given 2 spaced ~10⁸ cfu doses of CVD 103-HgR 10 days apart, and 8 control subjects, were challenged 1 month postvaccination with 10⁶ cfu of wild-type El Tor Ogawa strain E7946 (Bahrain epidemic isolate). In this small study, the attack rate for diarrhea with a total purge ≥ 1.0 l was 6/8 (75%) among controls versus 1/11 (6.8%) among vaccinees (vaccine efficacy 88% [95% CI, 18–98]) (p = 0.0063). In a subsequent volunteer challenge study, 19 volunteers who ingested a single ~10⁸ cfu dose of CVD 103-HgR either 10 (N = 10) or 30 days (N = 9) earlier were challenged, along with 8 controls, with 10⁶ cfu of wild-type El Tor Ogawa strain 3008. Diarrhea with a total purge ≥1.0 l was observed in 6 of 8 controls (75%) versus 3/19 vaccinees (15.8%) (vaccine efficacy 79% [95% CI, 36–93]) (p = 0.006). Results of these two El Tor Ogawa challenges were pooled to have enough subjects with ≥3.0 l purge to allow a statistical analysis of protection against moderate-to-severe El Tor Ogawa cholera. In total, 4/16 controls purged ≥3.0 l versus 0/30 vaccinees (100% VE) (p = 0.01).

4.4. SSVI high-dose formulation of CVD 103-HgR, Orochol E

4.4.1. Orochol E immunogenicity

Early clinical trials in pediatric populations living in impoverished, fecally contaminated conditions in North Jakarta, Indonesia, revealed that the serum vibriocidal response to a single ~5 × 10⁸ cfu dose of CVD 103-HgR, which stimulated high rates of seroconversion in North American and European adults, was poorly immunogenic in young children living in underprivileged conditions [46]. However, it was also found that by administering a single dose of the vaccine containing one log higher number of organisms (~5 × 10⁹ cfu), high rates of seroconversion could be achieved without reducing tolerability [45,46]. The minority of children who did not respond were, with few exceptions, ones with moderate or high baseline titers indicating prior exposure to V. cholerae O1 in this hyperendemic area. It was proposed that the environmental enteropathy that is the norm among children and adults living in squalid fecally contaminated conditions in developing countries is responsible for the diminished immune response to the standard-dose vaccine and that, accordingly, necessitates the high-dose formulation to achieve acceptable immunogenicity [46]. Levine [61] reviewed the clinical trials performed in multiple developing countries that affirmed this hypothesis and documented the acceptable immunogenicity and safety of the high-dose formulation, even in infants as young as 3 months of age [45–48,62–67]. The high-dose formulation for use in developing country populations was commercialized as Orochol E [50].

4.4.2. Field assessments of Orochol E efficacy

There were two field evaluations of the efficacy of Orochol E: one was a pre-licensure trial in North Jakarta [68] and the other was WHO’s field estimation of efficacy nested within the reactive mass immunization with CVD 103-HgR carried out on the island of Pohnpei in Micronesia during a large outbreak of El Tor Ogawa cholera [60].

4.4.2.1. North Jakarta field trial

The Jakarta field trial was undertaken in a densely populated area of the city where there was neither a reliable supply of potable water nor adequate sanitation and cholera was highly endemic (1–3 confirmed cases/10³ population/per year in the participating administrative units) before the trial. The field trial was designed before the ability of oral cholera vaccines to elicit powerful indirect protection was recognized. The investigators randomly allocated 67,508 enrolled participants aged 2–41 years to receive (1:1) vaccine or placebo; allocation was in blocks of two to have as many households as possible with at least one vaccinee and one placebo. An unexpected scarcity of cases of cholera following the administration of vaccine or placebo within these hyperendemic North Jakarta neighborhoods was swiftly noted. Only seven cases occurred within 6 months of vaccination, precluding assessment of short-term efficacy, and over 4 years of surveillance only 93 evaluable cases (43 vaccine, 50 placebo; vaccine efficacy = 14%).

The paucity of cholera cases may have been due to year-to-year secular trends. However, the cholera case drought observed after the vaccination in the first year of the trial remained so for 4 years within these previously high-risk communities. So, another plausible explanation is that the overall fall in disease burden was in fact related to the widespread use of vaccine in the highest risk portions of the population in a field trial whose design inadvertently optimized indirect protection. Indirect protection combined with direct protection could have diminished markedly the overall incidence of cholera in the participating households. The Jakarta field trial was not cluster randomized and was designed and executed before the pioneering report of Ali et al. [69] who brought attention to the powerful indirect protection that oral cholera vaccine can elicit. Ali et al. [69] reanalyzed data from the mid-1980s Matlab Bazar randomized, placebo-controlled field trial of two nonliving oral cholera vaccines (inactivated vibrios plus B subunit unit combination vs. inactivated vibrios vaccine alone [70]) and showed the impressive impact of indirect protection.

Rural Bangladesh communities where extended patrilineal-related families reside in baris allowed a reanalysis based on clusters (baris). As the proportion of vaccinated individuals in the clusters increased, the cholera incidence diminished not only among vaccinees but also among placebo recipients. At the lowest coverage level (<28% coverage), cholera incidence among vaccinees was 2.66/10³ vaccinees and dropped to 1.27/10³ vaccinees at the highest coverage level (>51% coverage). However, as vaccine coverage in the clusters increased, the cholera incidence also prominently fell among placebo recipients. While at the lowest level of vaccine coverage (<28% coverage), the incidence of cholera among placebo recipients was 7.01/10³ placebo recipients, where vaccine coverage was highest (>51% coverage), the cholera incidence among placebo recipients was only 1.47/10³ placebo recipients (only marginally higher than vaccinees in these clusters). In baris with the highest level of vaccine coverage, the point estimate of vaccine efficacy was only 14% (95% CI, −111–64%) [69]. Ali et al. [69] noted that high levels of vaccine coverage in individually randomized trials could bias estimates of vaccine efficacy in a downward direction and that analyses of oral cholera vaccines that do not assess indirect effects underestimate their potential public-health effects.

Longini et al. [71] used the Matlab Bazar field trial data to model the effect of different levels of vaccination coverage on reduction of disease burden in the target population and predicted that cholera transmission could be controlled in endemic areas with 50% coverage with oral cholera vaccines. They further predicted that vaccine coverage of only 30% would result in a 76% (95% CI, 44%–95%) reduction in incidence of cholera in the population covered.

Since the Jakarta study design would optimize indirect protective effects, moderate coverage with an effective vaccine could have drastically dropped the burden of disease in the field trial population, while making it difficult or impossible to demonstrate a significant point estimate of efficacy when comparing cholera incidence in vaccinees versus controls. Yet, despite inapparent vaccine efficacy, at the public health level, one would observe a marked overall reduction in cholera. This scenario may have ensued in North Jakarta.

4.4.2.2. Reactive vaccination during a cholera epidemic in Micronesia

A large epidemic struck the island of Pohnpei in Micronesia in 2000 [60]. While case fatality was kept low, the case load overwhelmed health-care facilities and the epidemic ravaged the island’s economy, as neighboring trade partners embargoed imports from Pohnpei [60]. The government decided to use oral cholera vaccine to control the epidemic and with the support of WHO 48,000 doses of Orochol E arrived in Pohnpei in September 2000. This was a historic cholera control response that represents the first reactive mass vaccination during an epidemic with an oral cholera vaccine. Moreover, various serendipitous opportunities, including a recently completed national census, computerized hospital records and impeccable vaccination registers during the mass immunization, allowed the team of WHO epidemiologists assisting in the outbreak to undertake a retrospective cohort analysis of vaccine efficacy.

Pohnpei health-care facilities that cared for cholera patients were mainly governmental and included Pohnpei Hospital and five dispensaries. The single private outpatient facility referred suspect cholera patients to Pohnpei Hospital which kept two computerized databases that recorded all inpatient admissions and outpatient/emergency room encounters, respectively. After V. cholerae O1 El Tor Ogawa had been laboratory confirmed, many cases of watery diarrhea listed as ‘cholera’ in the hospital registries were based on clinical diagnoses without undertaking bacteriologic confirmation.

The target population for vaccination included all Pohnpeians except children below age 2 years, pregnant women, persons with fever or diarrhea, or individuals taking antibiotics. The Public Health Department conducted the vaccination campaign and recorded in registers detailed demographic data on all vaccinees and their place and date of vaccination. Most Pohnpeians received Orochol E from 9 to 19 September.

Data from the 2000 nation-wide census that fortuitously had been completed before the cholera outbreak were used to define the size of a retrospective cohort that included all individuals living on Pohnpei proper who were older than 23 months of age in September 2000. This number closely resembled the population targeted for cholera vaccination on Pohnpei Island. For the retrospective vaccine efficacy evaluation, the subset of all cholera cases recorded after the vaccination campaign began was carefully matched with the vaccination registries. Suspect cholera cases recorded after an arbitrary interval of 10 days postvaccination (allowing some days for immunity to build) constituted the data for evaluation of Orochol E efficacy.

In total, 353 suspect cholera cases of all ages entered in the hospital databases after 19 September (10 days after the vaccination campaign began) were identified, of which 91 (26%) were laboratory-confirmed El Tor Ogawa; these included 59 cases among vaccine recipients and 294 non-vaccinated persons. Nine of the 59 vaccinated cases were excluded from analysis because onset of illness occurred within the 10-day postvaccination vulnerability window (n = 5); vaccination occurred after their cholera illness (n = 3) or vaccination status was insufficiently documented (n = 1), leaving 50 cases among appropriately vaccinated persons. Among the 294 non-vaccinated cases, 36 who were <2 years of age were removed, leaving 258 age-appropriate cases for analysis. Thus, in this retrospective cohort, which closely resembles the vaccine target population, the risk of developing cholera after vaccination was 16.47/10³ among the 15,664 unvaccinated individuals versus 3.43/10³ among the 14,587 vaccinated individuals, yielding an estimated vaccine efficacy of 79.2% (95% CI, 71.9–84.6) [60].

As with any assessment of vaccine efficacy that does not involve random allocation, the non-vaccinated Pohnpeians may have been inherently different and at higher risk than the Orochol E recipients. This limitation is applicable to any retrospective cohort or case/control assessment nested within a mass vaccination. Nevertheless, the field assessment during the Pohnpei outbreak provides additional evidence of the efficacy of CVD 103-HgR in preventing El Tor Ogawa cholera.

4.5. Discontinuation of manufacture of CVD 103-HgR by SSVI

SSVI experienced financial difficulties just before the Millennium and became Berna Biotech, which was acquired shortly thereafter by Crucell which stopped the manufacture of CVD 103-HgR and the license to the intellectual property reverted to the University of Maryland, Baltimore.

5. VAXCHORA

5.1. History

In 2009, PaxVax, a California-based vaccine biotechnology company, licensed rights to commercialize CVD 103-HgR from the University of Maryland and committed to an accelerated clinical development with the goal of obtaining approval of the vaccine by the FDA, thereby filling a void and making it the only cholera vaccine available for travelers from the USA. From the research master cell bank, a new Master Cell Bank and Working Cell Bank were produced under current Good Manufacturing Practices. The identity of the vaccine in the new GMP banks (PXVX0200) compared to Orochol was verified by genomic and microbiological characteristics and results of a Phase 1 clinical trial in US adults documented that vaccine prepared from the new Working Cell Bank was safe, well tolerated, and elicited serum vibriocidal responses [72].

5.2. Characteristics

The CVD 103-HgR product VAXCHORA manufactured by PaxVax, Inc. has the identical phenotypic and genomic properties as Orochol Berna (Section 3).

5.3. Formulation and storage

The VAXCHORA formulation of CVD 103-HgR for travelers from the USA contains ≥2 × 10⁸ cfu of lyophilized vaccine per single-dose sachet. CVD 103-HgR is grown in fermenters in medium containing casamino acids, yeast extract, mineral salt, and an antifoaming agent. Vaccine strain organisms are harvested by filtration, diafiltered and concentrated prior to adding a stabilization solution containing antitoxidant (ascorbic acid), a cryoprotectant (hydrolyzed casein from cow’s milk [Hy-Case SF]), stabilizer (sodium chloride), and another cryoprotectant (sucrose). The stabilized vibrios are lyophilized, milled, and blended with a dessicant-bulking agent (dried lactose) before filling the final material into sachets.

Buffer powder that accompanies each dose of vaccine, intended to neutralize gastric acid, is a blend of sodium bicarbonate, ascorbic acid (to counteract chlorine in water), and dried lactose (manufacturing flow aid). The buffer powder blend is filled into sachets.

5.3.1. Storage

The current initial VAXCHORA formulation of active component packets and buffer are stored at −25 to −15°C (−13–5°F).

5.4. Presentation and administration

A sachet-containing lyophilized vaccine and an accompanying sachet containing buffer powder are packaged into individual single-dose cartons for distribution. Packets do not require thawing prior to reconstitution. Reconstitution should be completed within 15 min of removing the carton containing the buffer (sachet #1) and the active vaccine component (sachet #2) from the freezer. Then, 100 mL of cold or room temperature (41–72°F; 5–22°C) purified bottled water is poured into a clean, disposable cup. Tap water, non-purified bottled water, other beverages, or other liquids should not be used. The top of the buffer sachet is opened and the buffer powder contents are put into the disposable cup. Effervescence will occur. Using a disposable stirrer, buffer and water should be stirred until the buffer component is completely dissolved. The top of the active component sachet is then opened and the lyophilized vaccine powder is put into the cup containing the buffer solution. The vaccine cocktail is finalized for oral administration by stirring for at least 30 s until the lyophilized vaccine disperses to form a slightly cloudy suspension that may contain some white particulates. The active component may not dissolve completely. VAXCHORA should be consumed within 15 min of reconstitution. The recipient should drink the full contents of the cup at once. If the sachets are reconstituted in the improper order, the vaccine must be discarded. Ideally, the vaccinee should fast for 60 min before and after ingesting the vaccine cocktail.

5.5. Safety and reactogenicity

Four randomized, placebo-controlled, clinical trials examined the safety of VAXCHORA:

1. Study 1 (NCT02094586, ClinicalTrials.gov), a multicenter, double-blind, randomized (8:1), placebo-controlled, 3-lot consistency trial conducted among adults 18–45 years of age in the USA and Australia;

2. Study 2 (NCT018955855, Clinical Trials.gov), a Phase 3 randomized (1:1), double-blind, placebo-controlled trial of the efficacy of a single dose of CVD 103-HgR live oral cholera vaccine in preventing cholera following challenge with V. cholerae 10 days or 3 months after vaccination, conducted at three US sites;

3. Study 3 (NCT01585181, Clinical Trials.gov), a Phase 1 randomized (5:1), double-blind, placebo-controlled study of the safety and immunogenicity of live oral CVD 103-HgR at two US sites;

4. Study 4 (NCT02100631, ClinicalTrials.gov), a multisite US Phase 3 randomized, double-blind, placebo-controlled study to assess immunogenicity and clinical acceptability of VAXCHORA in adults 46–64 years of age randomly allocated (3:1) to receive a single dose of vaccine or placebo.

These four trials collectively enrolled 3235 adults 18–64 years of age who were allocated to receive a single dose of CVD 103-HgR and 562 who got saline placebo ([N = 551] or lactose [N = 11)]. The mean age of participants overall was 32.5 years; 53.8% of participants were female; 67.1% were Caucasian, 27.3% were Black or African American, 1.8% Asian, 1.7% multiracial, 0.6% American Indian or Alaskan Native, 0.3% Native Hawaiian or Pacific Islander, and 1.3% were other; 9.3% were Hispanic or Latino.

5.5.1. Solicited adverse reactions

The most comprehensive dataset documenting the clinical acceptability of VAXCHORA comes from Study 1 (NCT02094586, ClinicalTrials.gov), the multicenter, double-blind, randomized (8:1), placebo-controlled 3-lot consistency trial conducted among adults 18–45 years of age in the USA and Australia. The safety analysis included 2789 CVD 103-HgR recipients and 350 placebo recipients. Solicited adverse reactions were recorded daily for 7 days following vaccination by 2734 of the 2789 vaccinees (98.0%) and by 343 of 350 placebo recipients (98.0%). The overall rate of diarrhea among vaccinees in Study 1 was 3.9% and was 1.2% among placebo recipients (p = 0.0079); 61.5% of the diarrheal complaints in vaccinees were mild. Fever was recorded in <1% of vaccinees. Studies 2 [59] and 3 [72] provided similar evidence of the live vaccine’s clinical acceptability in adults age 18–45 years, while Study 4 showed that the vaccine was well tolerated in adults age 45–64 years.

5.5.2. Serious adverse events

In a pooled analysis of the 4 clinical trials, 20 of 3235 VAXCHORA recipients (0.6%) and 3 of 562 placebo recipients (0.5%) reported a serious adverse event (SAE) within 6 months of vaccination. No SAEs were deemed vaccine related.

5.6. Immunogenicity

5.6.1. Serum-vibriocidal antibody responses to VAXCHORA

5.6.1.1. Three-lot consistency trial

Study 1 (NCT01895855, ClinicalTrials.gov) was a randomized, double-blind, saline placebo-controlled safety and immunogenicity study conducted in the USA and Australia in which 3146 subjects age 18–45 years not previously exposed to cholera were randomly allocated 8:1 to receive one dose of CVD 103-HgR or placebo. Mean age was 29.9 years; 45.2% were male; 68.3% were Caucasian, 25.6% Black, 2.0% Asian, 1.9% multiracial, and 4.0% were other; 10.0% were Hispanic or Latino. The Inaba vibriocidal antibody seroconversion rates were 93.5% [95% CI, 92.5–94.4%] in vaccine recipients and 4.2% [95% CI, 2.3–6.9%] in placebo recipients at 10 days postvaccination [73].

5.6.1.2. Challenge study to assess vaccine efficacy

Study 2 (NCT01895855, ClinicalTrial.gov) was a randomized, placebo-controlled study in which 197 healthy adult volunteers, 18–45 years of age in academic clinical research facilities in three US sites, were randomly allocated to receive a single dose of CVD 103-HgR (N = 95) or saline placebo (N = 102) [59]. The mean age of the participants was 31 years and 63% were males. Overall, 85 of 94 vaccinees (90.4%) manifested a ≥fourfold rise in serum Inaba vibriocidal antibody (seroconversion) following vaccination, including 84 of 93 (90.3% [95% CI, 82.4–95.5%]) who had serum from day 10 available for testing (1 vaccinee did not) had seroconversions evident by day 10 postvaccination, at which point the GMT peaked at 4313 (95% CI, 2873–6476); 2 of 102 placebo recipients (2.0% [95% CI, 0.2–6.9%]) seroconverted. There was no significant difference in vibriocidal antibody seroconversion or GMTs between blood group O or non-O vaccinees.

A subset of 134 enrollees participated in experimental challenge studies either 10 days (N = 35 vaccinees and 33 placebo recipients) or 3 months (N = 33 vaccinees and 33 placebo recipients) after vaccination to assess the efficacy of the vaccine in protecting against moderate (≥3.0 l total diarrheal purge) or severe (≥5.0 l total diarrheal purge) cholera. Subjects challenged were prioritized based on continued eligibility, availability, and blood type. There was no knowledge of their vibriocidal antibody response at the time of challenge. In total, 62 of 65 challenged vaccinees (91.2% [95% CI, 81.8–97.0%]) exhibited Inaba seroconversion by day 10 postvaccination [59]; one vaccinee did not have a day 10 serum specimen available for testing.

5.6.1.3. Study in adults 46–64 years of age

Study 4 (NCT02100631, ClinicalTrials.gov) was a randomized, double-blind, placebo-controlled study undertaken in the USA to assess the reactogenicity and immunogenicity of VAXCHORA. This study included 398 participants 46–64 years of age with no prior history of cholera infection or travel to a cholera-endemic area in the previous 5 years who were randomly allocated, 3:1, to receive a dose of CVD 103-HgR or placebo. The participants’ mean age was 53.8 years and 45.7% were males. Of the participants, 74.9% were Caucasian, 21.9% Black, and 3.4% were other; 7.5% were Hispanic or Latino. Among the 291 CVD 103-HgR recipients age 46–64 years whose sera were tested, the Inaba vibriocidal antibody seroconversion rate at 10 days postvaccination was 90.4% (95% CI, 86.4–93.5%) [73]. The serum Inaba vibriocidal antibody seroconversion rate at 10 days postvaccination among the adults 46–64 years of age in Study 4 was non-inferior to the seroconversion rate of Inaba vibriocidal antibody (93.5% [95% CI, 92.5–94.4%]) of the 2687 younger adults 18–45 years of age in Study 1.

5.6.2. Vibriocidal antibody seroconversion consistency in different populations

Table 3 summarizes seroconversion rates on day 10 following vaccination in several studies that involved different age groups of US and Australian adults. In each study, the overall rate of seroconversion was ≥90%, including in adults age 46–64 years, thereby showing remarkably consistent strong immunogenicity. Peak GMT was observed 10 days following ingestion of a single dose of VAXCHORA (Figure 1). Since serum vibriocidal antibody seroconversion was a strong correlate of protection in the challenge studies conducted at 10 days and 3 months postvaccination, the FDA accepted that serum vibriocidal seroconversion could be used to bridge immunogenicity and putative efficacy to other populations, such as pediatric hosts in industrialized countries. Other national regulatory agencies have also recently used non-inferiority of seroconversion of serum vibriocidal antibody to license Euvichol™, an inactivated V. cholerae vaccine closely resembling Shanchol, with both given as a two-dose regimen over 2 weeks [74]. Moreover, based on the non-inferiority data of serum vibriocidal antibody seroconversion, WHO pre-qualified Euvichol for procurement by United Nations agencies.

Figure 1. A plot of the kinetics of the serum Inaba vibriocidal antibody response (geometric mean titer [GMT] by day following immunization) of different groups in three different clinical trials who ingested a single dose of VAXCHORA. It is evident that in all vaccine groups the peak GMT is observed 10 days after immunization and that the titers fall progressively thereafter through day 180 post-vaccination but remain significantly above baseline. The Day 180 GMT remained significantly elevated above baseline GMT in the challenge, 3-lot consistency and older subject (age 46–64 years) studies (p = 0.001, p < 0.0001 and p < 0.001, respectively, calculated with paired t-test using log-transformed data)..

Table 3. Rates of seroconversion of serum vibriocidal antibody 10 days postvaccination with VAXCHORA or placebo among participants of different ages tested against different Vibrio cholerae O1 target strains.

	Study 1		Study 2		Study 4		Combined placebo recipients from all three studies
	3-lot Safety and immunogenicity study in 18–45 year olds in USA and Australia (NCT02094586, ClinicalTrials.gov)		18–45 year olds in USA (NCT01895855, ClinicalTrials.gov)		46–64-year olds in USA (NCT02100631)
V. cholerae O1 target strain for measuring vibriocidal antibody	No. serum pairs tested	% with ≥fourfold rises (95% CI)	No. serum pairs tested	% with ≥fourfold rises (95% CI)	No. serum pairs tested	% with ≥fourfold rises (95% CI)	No. serum pairs tested	% with ≥fourfold rises (95% CI)
Classical Inaba	2687	93.5% (92.5–94.4%)	93	90.3% (82.4–95.5%)	291	90.4% (86.4–93.5%)	533	3.0% (1.7–4.8%)
El Tor Inaba	–	NT*	93	91.4% (83.8–96.2%)	290	91.0% (87.1–94.1%)	198	4.5% (2.1–8.5%)
Classical Ogawa	–	NT*	93	87.1% (78.5–93.2%)	291	73.2% (67.7–78.2%)	198	2.5% (0.8–5.8%)
El Tor Ogawa	-	NT*	93	89.2% (81.1–94.7%)	290	71.4% (65.8–76.5%)	198	5.6% (2.8–9.7%)

*NT: Not tested.

In the VAXCHORA multisite challenge studies, 62 of 68 challenged vaccinees exhibited seroconversions [59]. Of the six challenged vaccinees who did not manifest seroconversions, four of six developed moderate-to-severe cholera (67%). In contrast, only 2 of the 62 vaccinees who seroconverted (3.2%) developed cholera (p = 0.00026 vs. non-seroconvertors) and their total diarrheal volume was just marginally above the 3-l purge cutoff that denotes moderate severity [59]. These data indicate that vibriocidal antibody seroconversion is a correlate of protection.

5.6.3. Duration of elevated serum vibriocidal antibody titers

Figure 1 plots the kinetics of the serum Inaba vibriocidal antibody titer following ingestion of a single dose of VAXCHORA. Peak GMT occurred at 10 days postvaccination. Although the titers in vaccinees from the three trials were considerably lower by day 180, the GMT remained significantly elevated compared to baseline and to placebo recipients. In vaccinees from industrialized countries, long-term monitoring of how long the serum vibriocidal titers remain above baseline plus monitoring of IgG B memory cells that recognize V. cholerae O1 lipopolysaccharide (LPS) antigens [75] may allow a correlation to be made with long-term protection conferred by CVD 103-HgR. This could be confirmed if some vaccinees agreed to participate in a challenge 1–3 years after vaccination analogous to the duration-of-protection study in the 1970s that rechallenged North American volunteers who had experienced experimental wild-type V. cholerae O1 cholera approximately 3 years earlier [22].

5.7. VAXCHORA efficacy

The efficacy of a single dose of VAXCHORA in protecting against cholera was demonstrated in experimental challenge studies wherein volunteers who were randomly allocated to receive a single dose of vaccine or placebo were challenged 10 days or 3 months after vaccination (Tables 1 and 2) [59]. The study protocol addressed the ability of the vaccine to prevent moderate (total diarrhea purge ≥3.0 l) and severe (total diarrheal purge ≥5.0 l) cholera. These are clinical levels of severity that could be life-endangering in the field in the absence of prompt and appropriate rehydration with water and electrolytes; 3.0 l approximates the total plasma volume and 5.0 l approximates the entire blood volume of a 70-kg adult. Moreover, the volunteer pool participating in the study was enriched in persons of blood group O, a strong host risk factor for the development of cholera gravis, to assure that the vaccine would protect such high-risk hosts [59]. As shown in Table 2, vaccine efficacy was 90% (95% CI, 61.7–100) when volunteers were challenged a mere 10 days after vaccination and was 80% (95% CI, 49.1–100) when subjects were challenged at 3 months [59].

6. Conclusion

VAXCHORA, the new PaxVax commercial formulation of CVD 103-HgR is well tolerated, elicits seroconversion of serum vibriocidal antibody (a correlate of protection) in circa 90% of industrialized country adults including subjects 18–64 years of age and protection is evident as early as 10 days following ingestion of the single dose. These admirable characteristics of the new formulation make it an attractive active immunoprophylactic against cholera for adult travelers to countries and regions where cholera transmission is occurring.

7. Expert commentary

The licensure of PaxVax CVD 103-HgR (VAXCHORA) by the FDA has reestablished the manufacture and commercial supply of this single-dose live oral vaccine, thereby providing a cholera vaccine for US travelers. Whereas several oral vaccines containing inactivated V. cholerae (alone or combined with B subunit of CT) given in a regimen of two doses spaced 2 weeks apart are licensed and used in other countries, none is licensed by the FDA and available to US travelers (Table 4). Shanchol and Euvichol are being increasingly used in developing countries to minimize seasonal epidemics and to control cholera outbreaks, while Dukoral is mainly utilized to prevent cholera in European travelers (Table 4). The US standard-dose formulation of VAXCHORA that contains ≥ 2 × 10⁸ cfu is intended to prevent cholera in the US travelers and in the future also to protect travelers from other industrialized countries who may be going to regions of active cholera transmission. Safety and immunogenicity studies to be initiated in 2017 will establish the safety and immunogenicity of the US formulation of VAXCHORA for children including pediatric travelers. The demonstration in challenge studies that seroconversion of serum vibriocidal antibody titers is a robust correlate of protection provides a means of bridging the seroconversion rate in children and elderly persons (≥65 years) to that of non-elderly adult subjects to imply likely protection.

Table 4. A comparison of some salient features of three licensed nonliving oral cholera vaccines and live oral CVD 103-HgR (VAXCHORA).

	Shanchol	Euvichol	Dukoral	VAXCHORA
Type of vaccine	Inactivated V. cholerae O1 (Inaba and Ogawa serotypes and classical and El Tor biotypes) and O139 bacteria	Inactivated V. cholerae O1 (Inaba and Ogawa serotypes and Classical and El Tor biotypes) and O139 bacteria	Inactivated V. cholerae O1 bacteria (Inaba and Ogawa serotypes and classical and El Tor biotypes) combined with plus B subunit of cholera toxin	Engineered attenuated V. cholerae O1 classical Inaba strain CVD 103-HgR used as a live oral vaccine
Route of administration	Oral	Oral	Oral	Oral
Immunization regimen	2 Doses given 2 weeks apart	2 Doses given 2 weeks apart	2 Doses given 2 weeks apart	Single dose
Requires buffering of gastric acid	No	No	Yes	Yes
Requires water for administration	No	No	Yes	Yes
Stimulates serum vibriocidal antibody	Yes	Yes	Yes	Yes
Stimulates cholera antitoxin	No	No	Yes	Yes
Protects immunologically naïve hosts with a single dose	No	No data; presumably not	No	Yes
Cold chain temperature requirements	2–8°C	2–8°C	2–8°C*	−25 to −15°C^†
Cold chain volume requirements in cm^3 per dose	16.8 cm^3	11 cm^3	As single dose: 271 cm^3 In 170-dose pack: 24 cm^3	NA^‡

*A volunteer challenge study with an early prototype B subunit/killed whole-cell combination vaccine did confer significant protection.

^†Studies are underway to develop a formulation that can be stored at 2–8°C, the storage conditions of the previous CVD 103-HgR commercial products Orochol, Mutacol, and Orochol E.

^‡The volume requirements of the VAXCHORA formulation that will be used for vaccinating populations in developing countries are not yet available. However, given the small size and thinness of the VAXCHORA double sachets, the cold chain storage requirements are expected to be modest.

Further studies are needed to expand knowledge of the characteristics and performance of the vaccine. In parallel, data are being gathered to render the cold chain requirements for VAXCHORA to be less stringent and to simplify storage of the vaccine.

One high priority is to measure the duration of protection conferred by a dose of CVD 103-HgR. Appropriately designed challenge studies in volunteers and a controlled field trial in an endemic area with extended follow-up would provide invaluable information to document the duration of protection. Ideally, results of an experimental challenge study of volunteers who were randomly allocated to receive CVD 103-HgR or placebo at least 12 months earlier would provide definitive evidence of protection in immunologically naïve enduring at least 1 year; successful protection upon challenge 18 or 24 months post-immunization would yield even more useful and compelling data but such challenge studies are logistically very difficult to complete and are very expensive. Alternatively, absent such data from challenge or field studies, since the FDA has accepted serum vibriocidal antibody seroconversion as the basis of bridging for evidence of protection of subjects who cannot be experimentally challenged, such as the elderly and children, one may propose that the longevity of serum vibriocidal antibody titers remaining at least twofold above the baseline, plus perhaps presence of IgG anti-LPS B memory cells, may be considered evidence of enduring protection.

The current VAXCHORA formulation must be kept frozen (−25 to −15°C) prior to use. Studies are underway to develop an improved formulation to allow storage at 2–8°C for up to 2 years, as was possible with the earlier commercial formulations (Orochol, Mutacol, and Orochol E). It is hoped that the improved formulation of VAXCHORA under development may be sufficiently stable out of a cold chain at ambient temperatures of 25–40°C for a period long enough (several days) to allow mass vaccinations in the field. Reducing cold chain stringency would markedly enhance the logistical practicality of vaccination with CVD 103-HgR.

The inactivated V. cholerae vaccines that do not contain B subunit and that have an immunization regimen of two doses administered 2 weeks apart have accumulated extensive experience in developing countries for the protection of adult and pediatric subjects where cholera is endemic or exhibits seasonal epidemics. These vaccines have also been employed occasionally in non-endemic populations where cholera has appeared in epidemic form. What is clear from these studies is that two doses of this type of vaccine provide ~65% protection that endures for up to 5 years, at least where high endemicity may boost immunity on a repetitive basis [2]. However, these vaccines confer a lower level of protection upon children <5 years of age, the population group in endemic areas that suffers the highest incidence of cholera [76]. Moreover, whereas significant protection was observed in a field trial in Kolkata among persons ≥5 years of age during the first year after vaccination, this was not so in children less than <5 years of age [76]. However, in ensuing years, the young Kolkata children exhibited protection, suggesting that the vaccine may have immunologically primed the young children to manifest boosts in immunity upon subsequent exposure to V. cholerae, thereby leading to enhancement of the primary vaccine-derived immunity [76].

Serum vibriocidal antibody responses have been used extensively to assess the immunity conferred by inactivated oral vaccines, as they have been with live oral vaccines. Euvichol was licensed by the Korean national regulatory authority based on non-inferiority in the rate of seroconversion compared to Shanchol in a head-to-head comparison [74]. Moreover, based on studies in Kolkata and Dhaka that documented rises in titer of serum vibriocidal antibodies following the first of two doses of Shanchol [77,78], it was proposed that this vaccine might protect following the ingestion of a single oral dose. To address this directly in a large efficacy trial in Dhaka, 204,700 subjects were individually randomized to receive 1 dose of oral inactivated vaccine or one dose of placebo [4]. Overall efficacy was 40% (95% CI, 11–60%). However, a single dose of vaccine did not confer significant protection in children <5 years of age (vaccine efficacy 16% [95% CI, −49–53%]). Thus, while a two-dose regimen of inactivated vibrio vaccine provides a moderate level of protection (~65%) among adults in endemic areas and a one-dose regimen confers ~56–63% short-term vaccine efficacy among adults living in a highly endemic area [4], a two-dose regimen is markedly less protective in children < 5 years of age [76] and a one-dose regimen did not confer significant protection in this age group (16% vaccine efficacy) [4]. Another recent case-cohort study that assessed vaccine effectiveness against severe cholera in an outbreak in a putatively immunologically primed population in South Sudan reported an unadjusted single-dose vaccine effectiveness of 80.2% (95% CI, 61.5–100.0). The authors opined that the very short period of surveillance (<2 months), the severity of cases detected and the presumed prior immunological priming of the population collectively accounted for the high estimate of short-term efficacy observed in this trial [79].

Single-dose live oral vaccine CVD 103-HgR may be useful in filling the population niche where the inactivated vaccines, given as either one or two doses, demonstrate markedly inferior performance, that is, children <5 years of age and immunologically naïve adults in non-endemic areas. However, in order to advance CVD 103-HgR to the point where it can fill this niche in protecting young children in endemic areas, multiple clinical trials and improvements in the vaccine formulation must be achieved. With respect to formulation, based on safety/immunogenicity trials with the earlier SSVI formulations of CVD 103-HgR, it is anticipated that a high-dose formulation containing ~10⁹ cfu will be required to achieve high rates of seroconversion in children and adults in developing countries where the prevalence of environmental enteropathy creates a relative barrier to oral immunization [46,47,61,62,66]. A recently completed preliminary trial that compared the standard-dose formulation (≥2 × 10⁸ cfu) versus a high-dose formulation (≥2 × 10⁹ cfu) of PaxVax CVD 103-HgR found the latter to be more immunogenic (ClinicalTrials.gov number, NCT02145377).

Additional clinical trials in developing countries are planned to assess the safety and immunogenicity of the high-dose formulation of PaxVax CVD 103-HgR in pediatric subjects including infants [48] and in HIV-positive subjects [80] and pregnant women. Finally, it will be important to undertake ethically appropriate post-licensure assessments of the efficacy and effectiveness of the high-dose formulation of PaxVax CVD 103-HgR in preventing cholera in the field.

Two field studies assessed the ability of Orochol E, the high-dose ~10⁹ cfu SSVI formulation of CVD 103-HgR, to prevent cholera in developing country populations. A double-blind, individual-randomized, placebo-controlled trial conducted in cholera-endemic North Jakarta, Indonesia (33,696 vaccinees, 33,812 placebo recipients) failed to show significant protection over 4 years of follow-up (14% vaccine efficacy) [68]. However, following vaccination, there was a marked fall in cholera cases in the study population overall, both vaccinees and placebo recipients, compared to the 4 years before the trial. So, an alternative explanation is that rather than a lack of efficacy against the endemic El Tor Ogawa strain, powerful indirect protection may have bolstered direct vaccine protection to diminish the overall incidence of cholera in controls as well as in vaccinees to the point efficacy where could not be demonstrated. The individual-randomized design of that trial with allocation blocks of 2 could have yielded such a result [68]. In their reanalysis of field trial of two inactivated oral cholera vaccines, Ali et al. [69] noted that as vaccine coverage increased, cholera incidence in the placebo group fell progressively, even as it did in vaccinees. When coverage reached ≥51%, cholera largely disappeared from both the vaccinated and placebo recipients, yet the point estimate of vaccine efficacy was only 14% [69]. Longini et al. [71] used those Bangladesh data to model vaccine-achievable control of cholera and predicted that in a population where facile transmission of cholera occurred, 30% coverage with a moderately efficacious vaccine would diminish by 76% (95% CI, 44–95%) the cholera incidence in the population covered.

The other assessment of the high-dose CVD 103-HgR formulation occurred during a cholera epidemic on Pohnpei Island, Micronesia, where WHO undertook a reactive single-dose mass immunization campaign with Orochol E [60]. Every individual who received a dose of vaccine was recorded, identities of all cholera cases in the post-vaccine campaign period were known and a census had been completed prior to the epidemic. Using these data in a retrospective cohort analysis, the WHO team calculated 79% vaccine efficacy (95% CI, 72–85%) in preventing El Tor Ogawa cholera under field conditions [60]. This was the first reactive immunization with an oral cholera vaccine to control epidemic cholera. Not until 2008 was oral inactivated cholera vaccine used for the first time in a reactive immunization during a cholera epidemic in Vietnam and a nested case/control study was undertaken to estimate the efficacy of the inactivated vaccine [81]. In the assessments of the efficacy of each of these reactive mass immunizations, vaccine was not randomly allocated and so vaccinees and non-vaccinated individuals could have differed in ways that influenced their risk of cholera. Also, only a portion of the cases during these outbreaks was culture confirmed, 29% in Pohnpei and ~50% in Vietnam. Despite these limitations, these field-based assessments of live oral CVD 103-HgR [60] and of Vietnamese oral killed vaccine [81] provide evidence of the protective efficacy of these vaccines when used in public health emergencies.

8. Five-year view

Peering into the future, over the next 5 years we envision that cholera will persist as a a public health problem in developing and early transitional countries, exacerbated by the migration of rural populations into urban areas where they often reside in settlements with ramshackle housing that lack potable water and sanitation. Living in such crowded unsanitary conditions fosters the facile transmission of cholera. Massive ‘virgin-soil’ outbreaks of cholera in Peru and Ecuador in 1991 and Haiti in 2010 exemplify the consequences of water-borne transmission of cholera in populations living without potable water and sanitation, even in the Western Hemisphere. With improved surveillance in sub-Saharan Africa, it is becoming increasingly evident that cholera transmission in that global region is far more common than was previously appreciated. To help control cholera in these less-developed settings, we envision increasingly wider use of oral cholera vaccines in developing countries with vaccines coming from a much larger and more diverse cholera vaccine stockpile than currently exists; an inventory of several types of cholera vaccines suitable for different epidemiological needs would enhance the flexibility and options available to public health practitioners committed to controlling cholera.

We expect that the oral vaccines based on inactivated vibrios will continue to be used to blunt the magnitude of seasonal epidemics of cholera where ‘hot spots’ of endemicity and seasonality patterns are well known. Such preemptive immunization should be carried out one or more months before the expected onset of the cholera season and with careful preparation even two doses can be administered in such preemptive situations. In contrast, when cholera breaks out in explosive fashion in immunologically unprimed populations, that is, ‘virgin-soil’ epidemics, which are often accompanied by higher than usual case fatality rates and widespread civic disruption, we envision that this will be an indication for the use of CVD 103-HgR and other live oral vaccines that can be administered as a single dose and can rapidly elicit protection within 10 days or less. Provided that appropriate clinical data supporting use in younger children are generated, we also foresee more programmatic use of CVD 103-HgR in endemic areas to immunize children <5 years of age, complementing the use of inactivated cholera vaccines that will be used in older subjects. It is also conceivable that the coadministration of powerful oral adjuvants with inactivated vibrio oral cholera vaccines may enhance the immunogenicity of the nonliving antigens to rival the vibriocidal responses elicited by live oral vaccine in immunologically unprimed subjects and allow protection with a single dose. Coadministration of mucosal adjuvants may also enhance the immunogenicity and efficacy of live oral cholera vaccines such as VAXCHORA.

Finally, we envision that the CVD 103-HgR formulation used in developing country populations in the future will contain ≥2 × 10⁹ cfu will be stable outside of the cold chain at tropical temperatures for an extended period (perhaps several weeks) and will not require water to administer the vaccine. We believe that improved vaccine presentations of this type that should be available in the future will revolutionize the administration of CVD 103-HgR and other live oral vaccines.

Key issues

• PaxVax re-established the manufacture and commercial supply of single-dose live oral cholera vaccine CVD 103-HgR under the trade name VAXCHORA. This vaccine was licensed by the FDA in June 2016.

• CVD 103-HgR fills the void of providing a cholera vaccine for U.S. travelers. The high level of protection of immunologically-naïve subjects against cholera as early as 10 days after vaccination (90% efficacy in a volunteer challenge study) shows characteristics that are particularly attractive for travelers to areas where cholera transmission is ongoing, as many travelers to such regions have short notice before they must depart.

• Development of an improved formulation are underway that would have less stringent cold chain requirements (2–8 °C) than the initial VAXCHORA formulation (−25 °C to −15 °C).

• Since serum vibriocidal antibody seroconversion is a correlate of protection, vibriocidal antibody seroconversion can be used to bridge efficacy data in young adults to other age groups such as the elderly and children, as long as there is non-inferiority of serum vibriocidal antibody seroconversion. Pediatric studies commence in 2017.

• Collective data from the current VAXCHORA product and from earlier Swiss Serum and Vaccine Institute/Berna CVD 103-HgR products show the vaccine’s ability to protect against cholera due to V. cholerae O1 of either Inaba or Ogawa serotype and either El Tor or Classical biotype. The VAXCHORA formulation elicits strong serum vibriocidal seroconversion irrespective of the serotype or biotype of the target V. cholerae O1 in the assay.

• A high-dose formulation of PaxVax CVD 103-HgR is expected to be used to immunize populations in developing countries where environmental enteropathy creates a barrier to oral immunization. Data from an initial comparison of the standard (≥ 2x10⁸ cfu) versus a high-dose (≥ 2x10⁹ cfu) formulation indicate superiority of the latter.

• The void that CVD 103-HgR can fill on the international scene is to immunize young children < 5 years of age, including infants as young as 3 months of age, with a single dose of vaccine (provided appropriate age-specific immunogenicity data are generated) as well as immunologically-naive adults. Whereas inactivated vibrio vaccines protect immunologically-primed adults with 2 doses given 2 weeks apart and provide them a lower level of protection with a single dose, those vaccines do not protect children age < 5 years with a single dose.

• For developing country use, improvements in the presentation of CVD 103-HgR are desirable that will diminish reliance on a cold chain and eliminate the need for mixing vaccine with water (or drastically diminish the volume of water required).

• The simplicity of CVD 103-HgR’s manufacture allows a relatively low cost-of-goods. Assuming future WHO prequalification can be achieved, this vaccine could help diversify the cholera vaccine stockpile by providing vaccine for virgin-soil epidemics.

• Single-dose VAXCHORA offers much promise as an alternative approach for prevention of cholera.

Declaration of interest

MM Levine is a co-inventor of live oral cholera vaccine strain CVD 103-HgR and co-holder of patents. Some future royalties on commercial sales will flow to the University of Maryland and will be available to him to dispense as he wishes (e.g., to support other research). WH Chen is a recipient of a grant from PaxVax that supported experimental cholera challenge studies. JB Kaper is a co-inventor of live oral cholera vaccine strain CVD 103-HgR and co-holder of patents. Some future royalties on commercial sales will flow to the University of Maryland and will be available to him to dispense as he wishes (e.g., to support other research). M Lock is employed by PaxVax as a biostatistician. L Danzig is employed by PaxVax as VP of Clinical Development & Medical Affairs. M Gurwith is employed by PaxVax as Chief Medical Officer.The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Additional information

Funding

The clinical trials of the PaxVax CVD 103-HgR formulations were fully supported by PaxVax, Inc. The genetic engineering and construction of attenuated V. cholerae O1 Classical Inaba vaccine strain was supported by NIH R01 grant AI19716 from the National Institute of Allergy and Infectious Diseases, NIH. The early experimental cholera challenge and re-challenge studies and early studies of vibriocidal antibody were supported by NIH research contracts N01 42553 and N01 45251 and a grant from the US Army Medical Research and Development Command (DAMA17-78-C-8011). Early clinical trials with the Swiss Serum and Vaccine Institute/Berna formulations of CVD 103-HgR were supported N01 AI 12666, N01 AI 45251, N01 62528, U01 35948 and AI 15096 from the National Institute of Allergy and Infectious Diseases, NIH and by a grant from the Consultative Group on Vaccine Development of the National Vaccine Program, USA.