Roles of adjuvant and route of vaccination in antibody response and protection engendered by a synthetic matrix protein 2-based influenza A virus vaccine in the mouse.

BACKGROUND: The M2 ectodomain (M2e) of influenza A virus (IAV) strains that have circulated in humans during the past 90 years shows remarkably little structural diversity. Since M2e-specific antibodies (Abs) are capable of restricting IAV replication in vivo but are present only at minimal concentration in human sera, efforts are being made to develop a M2e-specific vaccine. We are exploring a synthetic multiple antigenic peptide (MAP) vaccine and here report on the role of adjuvants (cholera toxin and immunostimulatory oligodeoxynucleotide) and route of immunization on Ab response and strength of protection. RESULTS: Independent of adjuvants and immunization route, on average 87% of the M2e-MAP-induced Abs were specific for M2e peptide and a variable fraction of these M2e(pep)-specific Abs (average 15%) cross-reacted with presumably native M2e expressed by M2-transfected cells. The titer of these cross-reactive M2e(pep-nat)-specific Abs in sera of parenterally immunized mice displayed a sigmoidal relation to level of protection, with EC50 of approximately 20 microg Ab/ml serum, though experiments with passive M2e(pep-nat) Abs indicated that serum Abs did not fully account for protection in parenterally vaccinated mice, particularly in upper airways. Intranasal vaccination engendered stronger protection and a higher proportion of G2a Abs than parenteral vaccination, and the strength of protection failed to correlate with M2e(pep-nat)-specific serum Ab titers, suggesting a role of airway-associated immunity in protection of intranasally vaccinated mice. Intranasal administration of M2e-MAP without adjuvant engendered no response but coadministration with infectious IAV slightly enhanced the M2e(pep-nat) Ab response and protection compared to vaccination with IAV or adjuvanted M2e-MAP alone. CONCLUSION: M2e-MAP is an effective immunogen as approximately 15% of the total M2e-MAP-induced Ab response is of desired specificity. While M2e(pep-nat)-specific serum Abs have an important role in restricting virus replication in trachea and lung, M2e-specific T cells and/or locally produced Abs contribute to protection in upper airways. Intranasal vaccination is preferable to parenteral vaccination, presumably because of induction of local protective immunity by the former route. Intranasal coadministration of M2e-MAP with infectious IAV merits further investigation in view of its potential applicability to human vaccination with live attenuated IAV.

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 microg/ml) even after two consecutive infections but increased to approximately 50 microg/ml after the third infection. Competition with free M2e peptide indicated that approximately 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 > or = 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 mug/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.

Enhancement of neutralizing activity of influenza virus-specific antibodies by serum components.

The role of serum components in enhancing virus neutralizing (VN) activity of influenza virus A/PR/8/34 hemagglutinin (HA)-specific MAbs in vitro was investigated. The degree of enhancement depended on the MAb's fine specificity and heavy chain isotype and on type of serum. Greatest enhancement (>100-fold) was seen with sera from immunodeficient mice that lacked serum immunoglobulin. At least two serum components were involved: C1q and a heat-resistant factor. C1q was mandatory for enhancement, and other components of the complement system were not required. C1q appeared to operate by improving MAb-mediated inhibition of virus attachment to host cells and was most effective with MAbs that inhibited virus attachment poorly on their own. The heat-resistant factor enhanced VN activity only in the presence of C1q and appeared to operate by enhancing VN activity at a post-attachment stage.

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.

Fine specificity and sequence of antibodies directed against the ectodomain of matrix protein 2 of influenza A virus.

The ectodomain of matrix protein 2 (M2e) has remained remarkably conserved amongst human influenza A viruses and is a target for Abs with protective activity. For these reasons, M2e is being investigated for its potential as a broadly protective influenza A virus vaccine. Here, we report on the fine specificity and sequence of seven M2e-specific mAbs isolated from three BALB/c mice after different immunization protocols. The mAbs recognized epitopes comprised within a 13aa long peptide corresponding to M2e(4-16). They originated from 4 distinct precursor B cells and showed a highly restricted variable (V) gene usage, in that their heavy chain V regions were all formed by the same V(H), D and J(H) gene segments and their light chain V regions made use of only two distinct Vkappa genes (Vkappa19-15/IGKV6-15 and Vkappa8-30/IGKV8-30; NCBI/IMGT annotation, respectively). The consensus sequence of the expressed V(H) genes belongs to the J558/HV1 family. It showed 96% identity with the BALB/c germline gene J558.n/IGHV1S137 and 100% identity with a V(H) gene expressed by several BALB/c B-1 B cells. This suggests that the consensus sequence is that of a functional BALB/c germline V(H) gene. The genetic restriction of this response may in part underlie the generally poor M2e-specific Ab response induced by infection.

Influenza type A virus escape mutants emerge in vivo in the presence of antibodies to the ectodomain of matrix protein 2.

The ectodomain of matrix protein 2 (M2e) of human influenza type A virus strains has remained remarkably conserved since 1918. Because M2e-specific immunity has been shown to decrease morbidity and mortality associated with influenza virus infection in several animal models and because natural infection and current vaccines do not appear to induce a good M2e-specific antibody (Ab) response, M2e has been considered as potential vaccine for inducing cross-reactive protection against influenza type A viruses. The high degree of structural conservation of M2e could in part be the consequence of a poor M2e-specific Ab response and thus the absence of pressure for change. To assess this possibility, we studied the course of infection in SCID mice in the presence or absence of passive M2e-specific monoclonal Abs (MAbs). We found that virus mutants with antigenic changes in M2e emerged in 65% of virus-infected mice treated with M2e-specific but not control MAbs. However, the diversity of escape mutants was highly restricted since only two types were isolated from 22 mice, one with a proline-to-leucine and the other with a proline-to-histidine interchange at amino acid position 10 of M2e. The implications of these findings for the use of M2e as a broadly protective vaccine are discussed.

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 muMT mice, termed muMT(-8), and tested them for ability to recover from infection. muMT(-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 muMT(-8) mice injected with normal spleen cells survived and resolved the infection. Transfer of BCR-transgenic spleen cells, which contained approximately 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.