Influenza A virus infection engenders a poor antibody response against the ectodomain of matrix protein 2

BACKGROUND: Matrix protein 2 (M2) is an integral tetrameric membrane protein of influenza A virus (IAV). Its ectodomain (M2e) shows remarkably little diversity amongst human IAV strains. As M2e-specific antibodies (Abs) have been shown to reduce the severity of infection in animals, M2e is being studied for its capability of providing protection against a broad range of IAV strains. Presently, there is little information about the concentration of M2e-specific Abs in humans. Two previous studies made use of ELISA and Western blot against M2e peptides and recombinant M2 protein as immunosorbents, respectively, and reported Ab titers to be low or undetectable. An important caveat is that these assays may not have detected all Abs capable of binding to native tetrameric M2e. Therefore, we developed an assay likely to detect all M2e tetramer-specific Abs. RESULTS: We generated a HeLa cell line that expressed full length tetrameric M2 (HeLa-M2) or empty vector (HeLa-C10) under the control of the tetracycline response element. These cell lines were then used in parallel as immunosorbents in ELISA. The assay was standardized and M2e-specific Ab titers quantified by means of purified murine or chimeric (mouse variable regions, human constant regions) M2e-specific Abs in the analysis of mouse and human sera, respectively. We found that the cell-based ELISA was substantially more effective than immobilized M2e peptide in detecting M2e-specific Abs in sera of mice that had recovered from repetitive IAV infections. Still, titers remained low (< 5 μg/ml) even after two consecutive infections but increased to ~50 μg/ml after the third infection. Competition with free M2e peptide indicated that ~20% of M2e-specific Abs engendered by infection reacted with M2e peptide. In humans presenting with naturally acquired influenza virus infection, 11 of 24 paired sera showed a ≥ 4-fold increase in M2e-specific Ab titer. The Ab response appeared to be of short duration as titers were very low (average 0.2 μg/ml) in all patients at onset of infection and in controls, in spite of evidence for previous exposure to IAV. CONCLUSION: The results provide convincing evidence that M2e-specific Ab-mediated protection is currently lacking or suboptimal in humans

Prospects for Universal Influenza Virus Vaccine

The current vaccination strategy against influenza A and B viruses is vulnerable to the unanticipated emergence of epidemic strains that are poorly matched by the vaccine. A vaccine that is less sensitive to the antigenic evolution of the virus would be a major improvement. The general feasibility of this goal is supported by studies in animal models that show that immunologic activities directed against relatively invariant viral determinants can reduce illness and death. The most promising approaches are based on antibodies specific for the relatively conserved ectodomain of matrix protein 2 and the intersubunit region of hemagglutinin. However, additional conserved determinants for protective antibodies are likely to exist, and their identification should be encouraged. Most importantly, infection and current vaccines do not appear to effectively induce these antibodies in humans. This finding provides a powerful rationale for testing the protective activity of these relatively conserved viral components in humans

Vaccination with M2e-Based Multiple Antigenic Peptides: Characterization of the B Cell Response and Protection Efficacy in Inbred and Outbred Mice

The extracellular domain of the influenza A virus protein matrix protein 2 (M2e) is remarkably conserved between various human isolates and thus is a viable target antigen for a universal influenza vaccine. With the goal of inducing protection in multiple mouse haplotypes, M2e-based multiple antigenic peptides (M2e-MAP) were synthesized to contain promiscuous T helper determinants from the Plasmodium falciparum circumsporozoite protein, the hepatitis B virus antigen and the influenza virus hemagglutinin. Here, we investigated the nature of the M2e-MAP-induced B cell response in terms of the distribution of antibody (Ab) secreting cells (ASCs) and Ab isotypes, and tested the protective efficacy in various mouse strains.Immunization of BALB/c mice with M2e-MAPs together with potent adjuvants, CpG 1826 oligonucleotides (ODN) and cholera toxin (CT) elicited high M2e-specific serum Ab titers that protected mice against viral challenge. Subcutaneous (s.c.) and intranasal (i.n.) delivery of M2e-MAPs resulted in the induction of IgG in serum and airway secretions, however only i.n. immunization induced anti-M2e IgA ASCs locally in the lungs, correlating with M2-specific IgA in the bronchio-alveolar lavage (BAL). Interestingly, both routes of vaccination resulted in equal protection against viral challenge. Moreover, M2e-MAPs induced cross-reactive and protective responses to diverse M2e peptides and variant influenza viruses. However, in contrast to BALB/c mice, immunization of other inbred and outbred mouse strains did not induce protective Abs. This correlated with a defect in T cell but not B cell responsiveness to the M2e-MAPs.Anti-M2e Abs induced by M2e-MAPs are highly cross-reactive and can mediate protection to variant viruses. Although synthetic MAPs are promising designs for vaccines, future constructs will need to be optimized for use in the genetically heterogeneous human population

Complement Component C1q Enhances the Biological Activity of Influenza Virus Hemagglutinin-Specific Antibodies Depending on Their Fine Antigen Specificity and Heavy-Chain Isotype

We have previously observed that selected influenza virus hemagglutinin (HA)-specific monoclonal antibodies (MAbs) with poor virus-neutralizing (VN) activity in vitro exhibited greatly enhanced VN activity in vivo after administration to SCID mice. The same Abs displayed improved VN activity also when tested in vitro in the presence of noninactivated serum from SCID mice. To identify Ab-dependent properties and serum components that contributed to enhancement of Ab activity, we screened a large panel of HA-specific MAbs for hemagglutination inhibition (HI) in the presence of noninactivated serum from naive mice (NMS). We found that HI activity was enhanced by NMS depending on the Ab’s fine specificity (antigenic region Cb/E > Ca/A,D > Sa,Sb/B), its heavy-chain isotype (immunoglobulin G2 [IgG2] > IgG3; IgG1 and IgM negative), and to some extent also on its derivation (primary response > memory response). On average, the HI activity of Cb/E-specific MAbs of the IgG2 isotype isolated from the primary response was enhanced by 20-fold. VN activity was enhanced significantly but less strongly than HI activity. Enhancement (i) was destroyed by heat inactivation (30 min, 56°C); (ii) did not require C3, the central complement component; (iii) was abolished by treatment of serum with anti-C1q; and (iv) could be reproduced with purified C1q, the binding moiety of C1, the first complement component. We believe that this is the first description of a direct C1q-mediated enhancement of antiviral Ab activities

Roles of CD4(+) T-Cell-Independent and -Dependent Antibody Responses in the Control of Influenza Virus Infection: Evidence for Noncognate CD4(+) T-Cell Activities That Enhance the Therapeutic Activity of Antiviral Antibodies

Previous studies have indicated that B cells make a significant contribution to the resolution of influenza virus infection. To determine how B cells participate in the control of the infection, we transferred intact, major histocompatibility complex class II (MHC-II)-negative or B-cell receptor (BCR)-transgenic spleen cells into B-cell-deficient and CD8(+) T-cell-depleted μMT mice, termed μMT(−8), and tested them for ability to recover from infection. μMT(−8) mice that received no spleen cells invariably succumbed to the infection within 20 days, indicating that CD4(+) T-cell activities had no significant therapeutic activity on their own; in fact, they were harmful and decreased survival time. Interestingly, however, they became beneficial in the presence of antiviral antibody (Ab). Injection of MHC-II((−/−)) spleen cells, which can provide CD4(+) T-cell-independent (TI) but not T-cell-dependent (TD) activities, delayed mortality but only rarely resulted in clearance of the infection. By contrast, 80% of μMT(−8) mice injected with normal spleen cells survived and resolved the infection. Transfer of BCR-transgenic spleen cells, which contained ∼10 times fewer virus-specific precursor B cells than normal spleen cells, had no significant impact on the course of the infection. Taken together, the results suggest that B cells contribute to the control of the infection mainly through production of virus-specific Abs and that the TD Ab response is therapeutically more effective than the TI response. In addition, CD4(+) T cells appear to contribute, apart from promoting the TD Ab response, by improving the therapeutic activity of Ab-mediated effector mechanisms