Coinfection with Streptococcus pneumoniae modulates the B cell response to influenza virus.

Pathogen-specific antibodies (Abs) protect against respiratory infection with influenza A virus (IAV) and Streptococcus pneumoniae and are the basis of effective vaccines. Sequential or overlapping coinfections with both pathogens are common, yet the impact of coinfection on the generation and maintenance of Ab responses is largely unknown. We report here that the B cell response to IAV is altered in mice coinfected with IAV and S. pneumoniae and that this response differs, depending on the order of pathogen exposure. In mice exposed to S. pneumoniae prior to IAV, the initial virus-specific germinal center (GC) B cell response is significantly enhanced in the lung-draining mediastinal lymph node and spleen, and there is an increase in CD4(+) T follicular helper (TFH) cell numbers. In contrast, secondary S. pneumoniae infection exaggerates early antiviral antibody-secreting cell formation, and at later times, levels of GCs, TFH cells, and antiviral serum IgG are elevated. Mice exposed to S. pneumoniae prior to IAV do not maintain the initially robust GC response in secondary lymphoid organs and exhibit reduced antiviral serum IgG with diminished virus neutralization activity a month after infection. Our data suggest that the history of pathogen exposures can critically affect the generation of protective antiviral Abs and may partially explain the differential susceptibility to and disease outcomes from IAV infection in humans. Importance: Respiratory tract coinfections, specifically those involving influenza A viruses and Streptococcus pneumoniae, remain a top global health burden. We sought to determine how S. pneumoniae coinfection modulates the B cell immune response to influenza virus since antibodies are key mediators of protection.

Cooperativity between CD8+ T cells, non-neutralizing antibodies, and alveolar macrophages is important for heterosubtypic influenza virus immunity.

Seasonal epidemics of influenza virus result in ∼36,000 deaths annually in the United States. Current vaccines against influenza virus elicit an antibody response specific for the envelope glycoproteins. However, high mutation rates result in the emergence of new viral serotypes, which elude neutralization by preexisting antibodies. T lymphocytes have been reported to be capable of mediating heterosubtypic protection through recognition of internal, more conserved, influenza virus proteins. Here, we demonstrate using a recombinant influenza virus expressing the LCMV GP33-41 epitope that influenza virus-specific CD8+ T cells and virus-specific non-neutralizing antibodies each are relatively ineffective at conferring heterosubtypic protective immunity alone. However, when combined virus-specific CD8 T cells and non-neutralizing antibodies cooperatively elicit robust protective immunity. This synergistic improvement in protective immunity is dependent, at least in part, on alveolar macrophages and/or other lung phagocytes. Overall, our studies suggest that an influenza vaccine capable of eliciting both CD8+ T cells and antibodies specific for highly conserved influenza proteins may be able to provide heterosubtypic protection in humans, and act as the basis for a potential "universal" vaccine.

Cell-intrinsic defects in the proliferative response of antiviral memory CD8 T cells in aged mice upon secondary infection.

Although previous studies have demonstrated delayed viral clearance and blunted effector T cell responses in aged mice during infection, memory CD8 T cells and especially secondary responses have received less attention. In this study, we show that modest differences in the number of memory CD8 T cells formed in aged versus young animals were associated with altered memory CD8 T cell differentiation. Aged immune mice had increased morbidity and mortality upon secondary viral challenge, suggesting changes in T cell immunity. Indeed, virus-specific memory CD8 T cells from aged mice showed substantially reduced proliferative expansion upon secondary infection using multiple challenge models. In addition, this defect in recall capacity of aged memory CD8 T cells was cell-intrinsic and persisted upon adoptive transfer into young mice. Thus, the poor proliferative potential of memory T cells and altered memory CD8 T cell differentiation could underlie age-related defects in antiviral immunity.

Pharmacologic activation of the innate immune system to prevent respiratory viral infections.

Drugs that can rapidly inhibit respiratory infection from influenza or other respiratory pathogens are needed. One approach is to engage primary innate immune defenses against viral infection, such as activating the IFN pathway. In this study, we report that a small, cell-permeable compound called 5,6-di-methylxanthenone-4-acetic acid (DMXAA) can induce protection against vesicular stomatitis virus in vitro and H1N1 influenza A virus in vitro and in vivo through innate immune activation. Using the mouse C10 bronchial epithelial cell line and primary cultures of nasal epithelial cells, we demonstrate DMXAA activates the IFN regulatory factor-3 pathway leading to production of IFN-ß and subsequent high-level induction of IFN-ß-dependent proteins, such as myxovirus resistance 1 (Mx1) and 2',5'-oligoadenylate synthetase 1 (OAS1). Mice treated with DMXAA intranasally elevate mRNA/protein expression of Mx1 and OAS1 in the nasal mucosa, trachea, and lung. When challenged intranasally with a lethal dose of H1N1 influenza A virus, DMXAA reduced viral titers in the lungs and protected 80% of mice from death, even when given at 24 hours before infection. These data show that agents, like DMXAA, that can directly activate innate immune pathways, such as the IFN regulatory factor-3/IFN-ß system, in respiratory epithelial cells can be used to protect from influenza pneumonia and potentially in other respiratory viral infections. Development of this approach in humans could be valuable for protecting health care professionals and "first responders" in the early stages of viral pandemics or bioterror attacks.

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.

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 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.

Pneumolysin expression by streptococcus pneumoniae protects colonized mice from influenza virus-induced disease.

The response to influenza virus (IAV) infection and severity of disease is highly variable in humans. We hypothesized that one factor contributing to this variability is the presence of specific respiratory tract (RT) microbes. One such microbe is Streptococcus pneumoniae (Sp) that is carried asymptomatically in the RT of many humans. In a mouse co-infection model we found that in contrast to secondary bacterial infection that exacerbates disease, Sp colonization 10 days prior to IAV protects from virus-induced morbidity and lung pathology. Using mutant Sp strains, we identified a critical role for the bacterial virulence factor pneumolysin (PLY) in mediating this protection. Colonization with the PLY-sufficient Sp strain induces expression of the immune-suppressive enzyme arginase 1 in alveolar macrophages (aMø) and correlates with attenuated recruitment and function of pulmonary inflammatory cells. Our study demonstrates a novel role for PLY in Sp-mediated protection by maintaining aMø as "gatekeepers" against virus-induced immunopathology.

Vaccination with M2e-based multiple antigenic peptides: characterization of the B cell response and protection efficacy in inbred and outbred mice.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSION/SIGNIFICANCE: 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.

Protective antiviral antibody responses in a mouse model of influenza virus infection require TACI.

Antiviral Abs, for example those produced in response to influenza virus infection, are critical for virus neutralization and defense against secondary infection. While the half-life of Abs is short, Ab titers can last a lifetime due to a subset of the Ab-secreting cells (ASCs) that is long lived. However, the mechanisms governing ASC longevity are poorly understood. Here, we have identified a critical role for extrinsic cytokine signals in the survival of respiratory tract ASCs in a mouse model of influenza infection. Irradiation of mice at various time points after influenza virus infection markedly diminished numbers of lung ASCs, suggesting that they are short-lived and require extrinsic factors in order to persist. Neutralization of the TNF superfamily cytokines B lymphocyte stimulator (BLyS; also known as BAFF) and a proliferation-inducing ligand (APRIL) reduced numbers of antiviral ASCs in the lungs and bone marrow, whereas ASCs in the spleen and lung-draining lymph node were surprisingly unaffected. Mice deficient in transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), a receptor for BLyS and APRIL, mounted an initial antiviral B cell response similar to that generated in WT mice but failed to sustain protective Ab titers in the airways and serum, leading to increased susceptibility to secondary viral challenge. These studies highlight the importance of TACI signaling for the maintenance of ASCs and protection against influenza virus infection.

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.

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.

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.

Virus-neutralizing activity mediated by the Fab fragment of a hemagglutinin-specific antibody is sufficient for the resolution of influenza virus infection in SCID mice.

Antibodies (Abs) contribute to the control of influenza virus infection in vivo by reducing progeny virus yield from infected cells (yield reduction [YR]) and by inhibiting progeny virus from spreading the infection to new host cells (virus neutralization [VN]). Previous studies showed that the infection could be resolved in severe combined immunodeficiency (SCID) mice by treatment with hemagglutinin (HA)-specific monoclonal antibodies (MAbs) that exhibit both VN and YR activities but not by MAbs that exhibited only YR activity. To determine whether virus clearance requires both activities, we measured the therapeutic activity of an HA-specific MAb (VN and YR) and its Fab fragment (VN) by intranasal (i.n.) administration to infected SCID mice. Immunoglobulin G (IgG) and Fab cleared the infection with i.n. 50% effective doses (ED(50)s) of 16 and 90 pmol, respectively. To resolve an established infection solely by VN activity, Fab must be present in the respiratory tract at an effective threshold concentration until all infected cells have died and production of virus has ceased. Because IgG and Fab had different half-lives in the respiratory tract (22 and 8 h, respectively) and assuming that both operated mainly or solely by VN, it could be estimated that clearance was achieved 24 h after Ab treatment when both reagents were present in the respiratory tract at approximately 10 pmol. This dose was approximately 200 times larger than the respiratory tract-associated Ab dose resulting from administration of the intraperitoneal ED(50) (270 pmol) of IgG. This indicated that our procedure of i.n. administration of Ab did not make optimal use of the Ab's therapeutic activity.

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 degrees 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.

Induction of influenza type A virus-specific resistance by immunization of mice with a synthetic multiple antigenic peptide vaccine that contains ectodomains of matrix protein 2.

Matix protein 2 (M2) is a transmembrane protein of influenza type A virus. It contains a 23 aa long ectodomain (M2e) that is highly conserved amongst human influenza type A viruses. M2e-specific antibodies have been shown to restrict virus growth in vitro and in vivo and thus have the potential of providing cross-reactive resistance to influenza type A virus infection. We attempted to induce M2e-specific protection with synthetic multiple antigen peptide (MAP) constructs that contained covalently linked M2e- and Th-determinant peptides. Mice, vaccinated twice by the intranasal (i.n.) route with adjuvanted M2e-MAPs exhibited significant resistance to virus replication in all sites of the respiratory tract. Compared to mice primed by two consecutive heterosubtypic infections, resistance was of similar strength in nasal and tracheal tissue but lower in pulmonary tissue. Importantly, the protection in M2e-MAP- and infection-immunized mice appeared to be mediated by distinct immune mechanisms. This suggests that stronger protection may be achievable by combining both protective activities.