Annual influenza vaccination is the major method of prophylaxis to protect against seasonal influenza viruses. Seasonal inactivated influenza virus vaccines commonly induce antibodies directed against the globular head domain of the major viral surface glycoprotein, the hemagglutinin (HA). These antibodies inhibit interactions between the virus and sialylated host cell receptors by sterically blocking the receptor binding site of the HA. Antibodies binding to the head domain are usually highly neutralizing and can readily be measured in the serum using the established hemagglutination inhibition (HI) assay. An HI titer of 40 is regarded as a relatively robust surrogate marker for protection against influenza virus infection Citation[1]. In contrast to seasonal influenza virus vaccines that contain human viruses, pre-pandemic vaccines containing avian H5, H7 or H9 strains are poorly immunogenic. Pandemic candidate vaccines induce poor HI antibody responses and a two dose regime is required either with a high antigen dose (90 μg HA) or with an immunogenic adjuvant Citation[2,3]. In the spring of 2013, more than 130 cases of human infections with a novel avian H7N9 virus strain were reported in China, many of them severe Citation[4]. H7N9 virus isolates appear to have acquired the ability to bind to the mammalian receptors in the upper respiratory tract, alpha-2.6 linked sialic acid Citation[5–7]. Patients have been treated with neuraminidase (NA) inhibitors, but rapid emergence of resistant mutants seems common Citation[8]. Since influenza virus vaccines are the best prophylactic measure against infection, the WHO developed and distributed H7N9 vaccine seed strains. Clearly, this provides a good opportunity to study surrogate correlates of protection, particularly as previous clinical trials with H7 virus vaccines showed apparent low immunogenicity and required high doses and/or an adjuvant to elicit detectable HI titers. H7N9 vaccines are expected to be similarly challenging. Importantly, H7 vaccines induced protection from morbidity and mortality in animal models despite no or low HI titers Citation[9]. Blocking of virus attachment to the host cell by HI antibodies might therefore not be the only mechanism of protection. Vaccines that exhibit low immunogenicity in terms of HI antibodies might induce other types of immunity that can offer protection such as antibodies to other regions of the HA. The most prominent example for non-HI active but protective immunity are antibodies that target the conserved, membrane proximal stalk domain of the HA and are able to bind and/or neutralize viruses of divergent strains within one subtype, across subtypes and even across the two phylogenetic groups of influenza virus HAs Citation[10,11]. These antibodies exhibit no HI activity but neutralize the virus by inhibiting the fusion of viral and endosomal membranes, by inhibiting the maturation of the HA and possibly by other mechanisms including antibody-dependent cell-mediated cytotoxicity (ADCC). Stalk-reactive antibodies are being investigated as a possible correlate of protection and universal influenza virus vaccines based on the induction of stalk-reactive antibodies are under development Citation[12–16]. Humans are usually exposed to the HA of H3 influenza viruses, which is phylogenetically related to H7 HA and shares conserved epitopes in the stalk domain. We are therefore already primed for these conserved epitopes and stalk-specific antibodies might be boosted upon H7 vaccination, refocusing the immune response from the H7 head (to which humans are naive) toward the stalk domain Citation[11,17]. Non-neutralizing antibodies against the HA (head- or stalk-binding) might contribute to the protection as well by employing mechanisms like ADCC but also by complement-mediated cytotoxicity and direct virus lysis by complement. Measurement of these antibodies employing recombinant HAs in ELISAs might correlate with protection from severe infection even in the absence of strongly neutralizing antibodies. This correlate could be evaluated in a passive transfer mouse challenge model, ideally in FcγR humanized mice.
Antibodies against the other major viral surface glycoprotein, the NA, have long been ignored, but recent reports show that NA-based vaccines are able to protect from disease as well – mainly by inhibiting the enzymatic activity of the NA Citation[18–20]. This neuraminidase inhibiting (NI) activity might be a critical factor in the absence of neutralizing HI active antibodies (e.g., in the case of H7 vaccines) but might also contribute to protection induced by seasonal influenza virus vaccines. Assays to measure NI are well established and should be further developed to define a correlate of protection. If a NA-specific correlate of protection can be established in humans, then an ensuing discussion about standardization of biologically active NA in inactivated vaccines will be needed.
The immune response induced after influenza virus infection and vaccination is multifaceted initially consisting of the innate response followed by the adaptive humoral (B cells) and T cellular responses, and there are probably multiple correlates of protection. Activated B cells called antibody secreting cell (ASC) produce antibodies and detailed analysis of these cells may shed more light on humoral correlates of protection. H7-specific ASC responses are elicited after H7 vaccination Citation[3,21], and the level of response was similar to that observed in immunologically naïve children after inactivated seasonal vaccination Citation[22], suggesting that these vaccines do not effectively activate B cells. The goal of vaccination is to elicit effective memory B cells (MBC), which can rapidly differentiate into plasmablasts secreting high affinity-specific IgG antibodies to rapidly neutralize the virus. Although an H7N7 live attenuated influenza vaccine (LAIV) was poorly immunogenic, subsequent boosting with an inactivated H7N7 vaccine induced a rapid antibody response 7 days post vaccination, suggesting the ability of at least live virus vaccines to induce long-term memory responses Citation[101]. LAIV provides protection from laboratory-confirmed influenza, but no clear correlate of protection has been defined. Early studies showed that LAIV efficiently elicited nasal wash IgA and that serum IgA correlated with the level of local IgA; however, nasal wash was the least sensitive measure for detecting an immune response to H7N7 LAIV. Clearly, the induction of local IgA at the portal of entry of influenza would provide a good first line of defense against infection reducing viral shedding and associated spread.
Furthermore, CD4+ T cells are important in the development of the adaptive immune response to influenza virus. Particularly, as influenza virus-specific CD4+ T cells correlate with protection from disease and reduce symptoms after experimental influenza virus infection in man Citation[23]. Importantly, the higher the total number of CD4+ T cells, the shorter the duration of illness. CD4+ T cells provide an early immunological correlate for subsequent antibody responses after vaccination with H5N1 vaccines Citation[24,25], with three- to fourfold increase in CD4+ T cells predicting increases in serum antibody after booster doses. CD4+ cells can also be divided into subsets according to the cytokines they secrete such as the main subsets of Th1 (IFN-γ, IL-2 and TNF-α) or Th2 (IL-4, IL-5 and IL-10). The Th1 cytokine, IL-2, was associated with increased immune responses after H7N1 vaccination in man Citation[3]. Th1 cells may play a role in mediating long-lived MBC responses Citation[26] pointing to the need to evaluate these responses in vaccine trials. In the elderly, Th2 cytokines are better indicators of vaccine-induced protection after seasonal vaccination than Th1 cytokines and cytokine responses should be measured to better understand their relevance in protection Citation[27]. An improved understanding of the multiple arms of the immune response after infection with H7N9 and correlation with clinical disease severity would greatly facilitate a deeper understanding of what provides effective immunity against influenza viruses in general. Ideally, human challenge of H7N9 vaccinated volunteers with, for example, a live attenuated influenza H7 vaccine strain would provide invaluable information on the mechanism of protection allowing definition of correlates of protection. Newly identified mechanisms of protection (e.g., stalk-reactive antibodies or anti-NA antibodies) that might also apply to vaccination with seasonal influenza viruses could be correlated with protection by re-evaluation of efficacy trials or household transmission studies where baseline (e.g., preseason) sera are available.
The emergence of H7N9 and development of candidate vaccines against these viruses provides a timely opportunity for the influenza community to re-evaluate the mechanisms of protection and develop novel surrogate markers for pandemic as well as seasonal influenza virus vaccines. We urgently need to conduct detailed kinetic studies of the humoral and cellular immune responses induced after vaccination bearing in mind that correlates of protection may vary with vaccine formulation. The limited number of clinical studies with H7 vaccines has shown evidence of poor immunogenicity and we must seize this opportunity to improve our understanding of immunological mechanisms and correlates of protection.
Financial & competing interests disclosure
F Krammer was supported by an Erwin Schrödinger fellowship (J3232) from the Austrian Science Fund (FWF). Work regarding correlates of (broad) protection at the Department of Microbiology, Icahn School of Medicine at Mount Sinai is supported by a Centers for Excellence for Influenza Research and Surveillance (CEIRS) grant (HHSN26620070010C to Peter Palese). R Cox is funded by the Ministry of Health and Care Services, Norway, the Norwegian Research Council Globvac (220670/H10) program, the European Union (Univax 601738) and Jebsen Centre. 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.
No writing assistance was utilized in the production of this manuscript.
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Website
- H5N1 and H7 LAIV-IAV Prime-Boost Studies. http://www.who.int/vaccine_research/meetings/Kanta_Subbarao.pdf