Hyperimmune bovine colostrum for treatment of GI infections

Abstract

Hyperimmune bovine colostrum (HBC), produced by vaccination of a cow during gestation, is rich in targeted immunoglobulins, and can be used to treat a variety of diseases. The published history of HBC use for treating gastrointestinal infections in humans has developed over the past several decades and demonstrates the promise of this type of therapeutic for GI infectious disease. HBC, or purified derivative products, have been used successfully for treatment or prevention of cryptosporidiosis, shigellosis, rotavirus, enterotoxigenic E. coli, and C. difficile infection (CDI). Given the positive results of previous studies using HBC for treatment of CDI, we have produced HBC with antibodies against the two most important virulence factors of C. difficile, TcdA and TcdB, using a novel recombinant vaccine. Our preliminary results demonstrate efficacy of the HBC product for treatment of CDI in the gnotobiotic piglet model, and warrant more thorough investigation. HBC may provide an effective treatment alternative to antibiotics, which can spare the normal gut microflora, and reduce rates of recurrence and antibiotic resistance.

Keywords: :

• hyperimmune bovine colostrum
• Clostridium difficile
• gastrointestinal infection
• diarrhea
• treatment
• prevention

Colostrum is the first milk produced by a lactating dairy cow after the birth of the calf. Although colostrum is typically rich in numerous immunoglobulins designed to protect the neonatal bovine from environmental pathogens, repeated immunization of a pregnant dairy cow may stimulate production of high levels of IgG against a specified antigen. This hyperimmune bovine colostrum (HBC), rich in targeted IgG, is unlike conventional antimicrobials as it does not disrupt the integrity of the gut microflora, nor will it potentially lead to the emergence of new antibiotic-resistant organisms. After collection, HBC may be used in liquid form in its entirety, or the immunoglobulin component may be purified and fractionated into a solid concentrate. HBC has been investigated as a passive immunotherapeutic agent against a wide variety of enteric microorganisms, Clostridium difficile among them. Here we provide a historical review of relevant literature investigating the use of HBC or derivative products for the prevention and treatment of gastrointestinal (GI) infections in humans, with a focus on C. difficile and our recent work to evaluate HBC for treatment.

Perhaps the earliest published accounts of the use of HBC for the treatment of GI disease in human patients are for cryptosporidiosis. Cryptosporidium species generally cause self-limiting diarrhea in healthy individuals, but can cause chronic, wasting diarrhea in malnourished and immunocompromised populations. Cryptosporidiosis was recognized as an important gastrointestinal infection in HIV-infected humans in the 1980s. Tzipori et al. described effective treatment of cryptosporidiosis in immunocompromised children and adults with HBC administered orally.^1,2 Using immunoglobulin concentrate derived from HBC, Greenberg et al. showed that the powdered form, but not capsule form of the concentrate, was effective at treating diarrhea in C. parvum-infected AIDS patients.³ HBC was also found effective in the prevention of cryptosporidiosis in healthy volunteers when administered before challenge with C. parvum spores.⁴

Shigellosis, caused by various Shigella species, remains a common cause of diarrhea in young children in developing countries, in certain communal care settings, and in travelers and military personnel. While antibiotics are the standard of treatment, immunological approaches, including HBC, have been investigated for both treatment and prevention. Tacket et al. investigated prophylactic efficacy of immunoglobulin concentrate from HBC in healthy adult volunteers and found that the product significantly reduced the incidence of diarrhea resulting from challenge with S. flexneri.⁵ When HBC was investigated as a treatment for shigellosis in children, however, it was found to be ineffective, with no difference in diarrhea between treated and untreated children.⁶

Helicobacter pylori causes gastric infection in children and adults, and is especially prevalent in children in developing countries, where it is associated with chronic malnutrition and diarrhea. Opekun et al. evaluated healthy, adult H. pylori-infected patients who were given an oral suspension of an HBC cocktail with several IgG isotypes. Patients were assessed based on a urea breath test or a histological H. pylori count; results indicated that HBC had no significant effect on either measure.⁷ In a double-blind study, H. pylori-positive infants were administered either HBC or a colostrum placebo from a non-immunized cow for 30 days, but HBC was also ineffective in this population, as none of the infants tested negative by the urea breath test after the treatment.⁸ HBC may not contain sufficient concentrations of immunoglobulin to successfully treat the intracellular stage of H. pylori infection, since this study used the highest practical dose.⁸

Rotavirus is the primary cause of nonbacterial, acute diarrhea in infants and young children six months to two years of age in both developed and developing countries. No effective vaccine is available to date, therefore reduction of duration and severity of symptoms in infants is an important public health concern. Hilpert et al. treated infants hospitalized with acute diarrhea with anti-rotavirus immunoglobulin concentrate without a significant decrease in duration of diarrhea or excretion of virus.⁹ However, Mitra et al. showed a decrease in both duration of diarrhea and reduction in stool output when treating rotavirus-infected infants with HBC.¹⁰ In addition, two groups tested the efficacy of passive immunization of healthy infants with anti-rotavirus HBC and demonstrated a reduction in incidence and duration of diarrhea in treated infants.^11,12

Enterotoxigenic Escherichia coli (ETEC) is a common bacterial pathogen associated with traveler?s diarrhea and infant bacterial diarrhea in developing countries. Current preventative treatments are limited and not ideal as long-term solutions. Several groups have used either HBC or milk concentrate IgG to passively immunize healthy adults against ETEC. Tacket et al. showed that anti-E. coli IgG was effective at preventing diarrhea when given prior to challenge with ETEC,¹³ and a study by Freedman et al. had similar results.¹⁴ In a study of 90 healthy adults, Otto et al. used a tablet form of HBC to successfully prevent diarrhea development in 93% of treated volunteers receiving the highest dose, and this is the basis for the commercially available tablets, sold as Travelan in Australia.¹⁵ In addition, Tawfeek et al. tested the efficacy of anti-ETEC HBC antibodies in preventing diarrhea in infants, and found a significant reduction in incidence and duration of diarrhea in HBC-fed infants compared with formula-fed, but not breast-fed, infants.¹⁶

Clostridium difficile is a spore-forming, Gram-positive bacterium that has emerged as a leading cause of infectious diarrhea. C. difficile-associated diarrhea primarily affects aged patients as a sequela of antimicrobial therapy, often during hospitalization.¹⁷C. difficile accounts for up to 25% of cases of antibiotic-associated diarrhea,¹⁸ and nosocomial C. difficile infection (CDI) is linked to high morbidity, resulting in prolonged patient stays and subsequent increases in healthcare costs. American acute-care providers likely incur C. difficile-related medical expenditures in excess of four billion dollars annually,¹⁹ while the cost to long-term care facilities is largely unknown. In addition to diarrhea, CDI may generate fulminant disease manifested by pseudomembranous colitis, toxic megacolon, and death. C. difficile contributed to approximately 14,000 deaths in 2007,²⁰ and fatalities are rising, especially among the elderly.

Standard treatment for CDI is antimicrobial therapy; vancomycin, metronidazole, and more recently fidaxomicin, are typically employed.²¹ An undesirable consequence of these drugs is disruption of the ordinary microbial inhabitants of the intestinal tract. Disturbance of normal colonic microflora is a primary factor in the development of CDI and its recurrence.¹⁷ Despite aggressive antibiotic therapy, up to 35% of patients treated for CDI experience recurrent disease,²² and many have multiple relapses, resulting in significant debilitation and requiring months, or possibly years, of costly treatment. A single case of recurrent CDI in the US may cost up to $18,000 to treat, including extended hospitalization.²³ With the emergence of more virulent C. difficile strains exhibiting increased antibiotic resistance, CDI has become more refractory to conventional treatment, precipitating additional relapses following antimicrobial use.^24,25 In the past two decades, mounting evidence indicates a rise in the prevalence, incidence, and severity of CDI in North America,^26,27 necessitating the exploration of potential alternative therapeutic measures.

The use of HBC against CDI has produced promising preliminary results. Anti-C. difficile HBC was shown to be effective in inhibiting adhesion of C. difficile bacteria to enterocyte-like caco-2 cells in vitro.²⁸ In addition, concentrated HBC IgG was effective at neutralizing TcdA and TcdB in a tissue culture assay.²⁹ This same group was able to demonstrate that this HBC was effective at protecting hamsters against CDI, particularly when combined with Enfamil milk replacer. They also demonstrated that the HBC-IgG survived passage through healthy adult human gastrointestinal tracts, with the most IgG surviving and retaining neutralization activity when protected by enteric capsules or given with sodium bicarbonate and omeprazole.^30,31 Anti-C. difficile HBC has also been as effective at treating mild to moderate recurring CDI as metronidazole.^32,33

Since previous work with HBC as a treatment for CDI has shown positive results as described above, we performed a pilot study to evaluate the efficacy of an HBC produced at our institution. This follows our work to develop an effective recombinant vaccine against the C. difficile toxins, TcdA and TcdB.³⁴ Using this novel vaccine product, we immunized a pregnant Holstein dairy cow to generate colostrum with high titers of anti-TcdA and anti-TcdB antibodies. Using the gnotobiotic piglet model of CDI we previously developed at our institution, we evaluated the efficacy of the anti-C. difficile HBC for treatment,³⁵ comparing either the HBC or non-immune control colostrum in piglets.

Our preliminary trial using HBC for treatment of CDI in piglets suggests that our HBC product has the ability to prevent development of severe, fatal disease, and it reduced or resolved diarrhea, similar to previous HBC products for other GI infectious diseases. While further investigation is warranted, this initial study indicates that this HBC is an effective treatment for CDI and could be evaluated for use in human patients in the future. We plan to further evaluate the product in the piglet model, including a freeze-dried, powdered, or encapsulated form that would be shelf-stable and practical for easy oral administration.

CDI, which is commonly acquired following antibiotic use in a healthcare setting, poses a mounting health threat to an aging population. HBC has shown potential as an effective therapy against a variety of enteric pathogens, including C. difficile (Table 1). In this mini-review of HBC products, 14 of 18 (77%) of studies demonstrated that HBC or derivative products were effective for the treatment or prevention of GI infectious disease (Table 1). Treatment of CDI patients with HBC containing toxin?specific antibodies presents an alternative to conventional antimicrobials by providing an immunoglobulin-driven mechanism for eradication of disease. The use of HBC likely leaves the normal gut microflora intact, and also eliminates the looming specter of antibiotic resistance. Successful development of HBC as a novel immunotherapeutic agent could revolutionize current management of CDI, yielding improved outcomes for patients and bolstering public health. Though additional studies are needed before definitive conclusions may be drawn, hyperimmune bovine colostrum may be the advantage the medical community is seeking for the treatment of infections caused by Clostridium difficile.

Table 1. Hyperimmune bovine colostrum products for treatment and prevention of gastrointestinal infectious disease in humans

Disease	Reference	HBC product	Patients	Outcome
Cryptosporidiosis	Tzipori 1986	whole HBC^A	immunocompromised children and adults	resolved diarrhea
	Tzipori 1987	whole HBC		resolved diarrhea
	Greenberg 1996	Ig concentrate^B	infected adult AIDS	resolved diarrhea
	Okhuysen 1998	Ig concentrate	healthy challenged adults	prevented diarrhea
Shigellosis	Tacket 1992	Ig concentrate	healthy challenged adults	prevented diarrhea
	Ashraf 2001	whole HBC	infected children	ineffective
H. pylori	Opekun 1999	purified IgG^C	infected adults	ineffective
	Casswall 1998	whole HBC	infected infants	ineffective
Rotavirus	Hilpert 1987	Ig concentrate	infected infants	ineffective
	Mitra 1995	whole HBC	infected infants	reduced diarrhea
	Davidson 1989	whole HBC	healthy infants	reduced incidence of diarrhea
	Turner 1993	Ig concentrate	healthy infants	reduced incidence of diarrhea
Enterotoxigenic E. coli	Tacket 1988	purified IgG	healthy challenged adults	prevented diarrhea
	Freedman 1998	purified IgG	healthy challenged adults	prevented diarrhea
	Otto 2011	HBC tablets^D	healthy challenged adults	prevented diarrhea
	Tawfeek 2003	Ig concentrate	healthy formula-fed infants	reduced incidence of diarrhea^E
C. difficile	Mattila 2008	purified IgG	infected adults	resolved diarrhea
	Numan 2007	purified IgG	recovered adults^F	prevented relapse

^A unaltered, liquid HBC was administered; ^B immunoglobulins in the HBC were concentrated and administered in a dry form; ^C IgG against the desired antigen was purified from HBC and administered in a dry form; ^D whole HBC was dried and formed into tablets for administration; ^E diarrhea was only reduced in comparison with formula-fed, but not breast-fed, infants; ^F adult patients with resolved diarrhea after antibiotic treatment.

Abbreviations:
HBC	=	hyperimmune bovine colostrum
CDI	=	Clostridium difficile infection
IgG	=	Immunoglobulin G
ETEC	=	enterohemorrhagic Escherichia coli
TcdA	=	C. difficile toxin A
TcdB	=	C. difficile toxin B

Acknowledgments

We give special thanks to our animal care technicians, Patricia Boucher and Rachel Nieminen, who cared for all the piglets and cow used in the HBC experiment at our institution.

Submitted

02/01/13

Accepted

02/22/13

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.