Nayana Prabhu

Monoclonal Antibodies against the Fusion Peptide of Hemagglutinin Protect Mice from Lethal Influenza A Virus H5N1 Infection

ABSTRACT The HA2 glycopolypeptide (gp) is highly conserved in all influenza A virus strains, and it is known to play a major role in the fusion of the virus with the endosomal membrane in host cells during the course of viral infection. Vaccines and therapeutics targeting this HA2 gp could induce efficient broad-spectrum immunity against influenza A virus infections. So far, there have been no studies on the possible therapeutic effects of monoclonal antibodies (MAbs), specifically against the fusion peptide of hemagglutinin (HA), upon lethal infections with highly pathogenic avian influenza (HPAI) H5N1 virus. We have identified MAb 1C9, which binds to GLFGAIAGF, a part of the fusion peptide of the HA2 gp. We evaluated the efficacy of MAb 1C9 as a therapy for influenza A virus infections. This MAb, which inhibited cell fusion in vitro when administered passively, protected 100% of mice from challenge with five 50% mouse lethal doses of HPAI H5N1 influenza A viruses from two different clades. Furthermore, it caused earlier clearance of the virus from the lung. The influenza virus load was assessed in lung samples from mice challenged after pretreatment with MAb 1C9 (24 h prior to challenge) and from mice receiving early treatment (24 h after challenge). The study shows that MAb 1C9, which is specific to the antigenically conserved fusion peptide of HA2, can contribute to the cross-clade protection of mice infected with H5N1 virus and mediate more effective recovery from infection.

Lung CD103+ Dendritic Cells Efficiently Transport Influenza Virus to the Lymph Node and Load Viral Antigen onto MHC Class I for Presentation to CD8 T Cells

The uptake, transport, and presentation of Ags by lung dendritic cells (DCs) are central to the initiation of CD8 T cell responses against respiratory viruses. Although several studies have demonstrated a critical role of CD11blow/negCD103+ DCs for the initiation of cytotoxic T cell responses against the influenza virus, the underlying mechanisms for its potent ability to prime CD8 T cells remain poorly understood. Using a novel approach of fluorescent lipophilic dye-labeled influenza virus, we demonstrate that CD11blow/negCD103+ DCs are the dominant lung DC population transporting influenza virus to the posterior mediastinal lymph node as early as 20 h postinfection. By contrast, CD11bhighCD103neg DCs, although more efficient for taking up the virus within the lung, migrate poorly to the lymph node and remain in the lung to produce proinflammatory cytokines instead. CD11blow/negCD103+ DCs efficiently load viral peptide onto MHC class I complexes and therefore uniquely possess the capacity to potently induce proliferation of naive CD8 T cells. In addition, the peptide transporters TAP1 and TAP2 are constitutively expressed at higher levels in CD11blow/negCD103+ DCs, providing, to our knowledge, the first evidence of a distinct regulation of the Ag-processing pathway in these cells. Collectively, these results show that CD11blow/negCD103+ DCs are functionally specialized for the transport of Ag from the lung to the lymph node and also for efficient processing and presentation of viral Ags to CD8 T cells.

Development of epitope-blocking ELISA for universal detection of antibodies to human H5N1 influenza viruses.

Background Human infections with highly pathogenic H5N1 avian influenza viruses have generally been confirmed by molecular amplification or culture-based methods. Serologic surveillance has potential advantages which have not been realized because rapid and specific serologic tests to detect H5N1 infection are not widely available. Methodology/Principal Findings Here we describe an epitope-blocking ELISA to detect specific antibodies to H5N1 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (5F8) that binds to an epitope comprising amino acid residues 274–281 (CNTKCQTP) in the HA1 region of H5 hemagglutinin. Database search analysis of publicly available sequences revealed that this epitope is conserved in 100% of the 163 H5N1 viruses isolated from humans. The sensitivity and specificity of the epitope-blocking ELISA for H5N1 were evaluated using chicken antisera to multiple virus clades and other influenza subtypes as well as serum samples from individuals naturally infected with H5N1 or seasonal influenza viruses. The epitope-blocking ELISA results were compared to those of hemagglutinin inhibition (HI) and microneutralization assays. Antibodies to H5N1 were readily detected in immunized animals or convalescent human sera by the epitope-blocking ELISA whereas specimens with antibodies to other influenza subtypes yielded negative results. The assay showed higher sensitivity and specificity as compared to HI and microneutralization. Conclusions/Significance The epitope-blocking ELISA based on a unique 5F8 mAb provided highly sensitive and 100% specific detection of antibodies to H5N1 influenza viruses in human sera.

Gastrointestinal Delivery of Baculovirus Displaying Influenza Virus Hemagglutinin Protects Mice against Heterologous H5N1 Infection

ABSTRACT The recent outbreaks of influenza A H5N1 virus in birds and humans have necessitated the development of potent H5N1 vaccines. In this study, we evaluated the protective potential of an immediate-early promoter-based baculovirus displaying hemagglutinin (BacHA) against highly pathogenic avian influenza (HPAI) H5N1 virus infection in a mouse model. Gastrointestinal delivery of BacHA significantly enhanced the systemic immune response in terms of HA-specific serum IgG and hemagglutination inhibition (HI) titers. In addition, BacHA vaccine was able to significantly enhance the mucosal IgA level. The inclusion of recombinant cholera toxin B subunit as a mucosal adjuvant along with BacHA vaccine did not influence either the systemic or mucosal immunity. Interestingly, an inactivated form of BacHA was able to induce only a negligible level of immune responses compared to its live counterpart. Microneutralization assay also indicated that live BacHA vaccine was able to induce strong cross-clade neutralization against heterologous H5N1 strains (clade 1.0, clade 2.1, and clade 8.0) compared to the inactivated BacHA. Viral challenge studies showed that live BacHA was able to provide 100% protection against 5 50% mouse lethal doses (MLD50) of homologous (clade 2.1) and heterologous (clade 1) H5N1. Moreover, histopathological examinations revealed that mice vaccinated with live BacHA had only minimal bronchitis in lungs and regained their body weight more rapidly postchallenge. Furthermore, immunohistochemistry results demonstrated that the live BacHA was able to transduce and express HA in the intestinal epithelial cells in vitro and in vivo. We have demonstrated that recombinant baculovirus with a white spot syndrome virus (WSSV) immediate-early promoter 1 (ie1) acted as a vector as well as a protein vaccine and will enable the rapid production of prepandemic and pandemic vaccines without any biosafety concerns.

Combination Therapy Using Chimeric Monoclonal Antibodies Protects Mice from Lethal H5N1 Infection and Prevents Formation of Escape Mutants

Background Given that there is a possibility of a human H5N1 pandemic and the fact that the recent H5N1 viruses are resistant to the anti-viral drugs, newer strategies for effective therapy are warranted. Previous studies show that single mAbs in immune prophylaxis can be protective against H5N1 infection. But a single mAb may not be effective in neutralization of a broad range of different strains of H5N1 and control of potential neutralization escape mutants. Methods/Principal Findings We selected two mAbs which recognized different epitopes on the hemagglutinin molecule. These two mAbs could each neutralize in vitro escape mutants to the other and in combination could effectively neutralize viruses from clades 0, 1, 2.1, 2.2, 2.3, 4, 7 and 8 of influenza A H5N1 viruses. This combination of chimeric mAbs when administered passively, pre or post challenge with 10 MLD50 (50% mouse lethal dose) HPAI H5N1 influenza A viruses could protect 100% of the mice from two different clades of viruses (clades 1 and 2.1). We also tested the efficacy of a single dose of the combination of mAbs versus two doses. Two doses of the combination therapy not only affected early clearance of the virus from the lung but could completely prevent lung pathology of the H5N1 infected mice. No escape variants were detected after therapy. Conclusions/Significance Our studies provide proof of concept that the synergistic action of two or more mAbs in combination is required for preventing the generation of escape mutants and also to enhance the therapeutic efficacy of passive therapy against H5N1 infection. Combination therapy may allow for a lower dose of antibody to be administered for passive therapy of influenza infection and hence can be made available at reduced economic costs during an outbreak.

Influenza A Virus Infection Results in a Robust, Antigen-Responsive, and Widely Disseminated Foxp3+ Regulatory T Cell Response

ABSTRACT Foxp3+ CD4+ regulatory T cells (Tregs) represent a highly suppressive T cell subset with well-characterized immunosuppressive effects during immune homeostasis and chronic infections, although the role of these cells in acute viral infections is poorly understood. The present study sought to examine the induction of Foxp3+ CD4+ Tregs in a nonlethal murine model of pulmonary viral infection by the use of the prototypical respiratory virus influenza A. We establish that influenza A virus infection results in a robust Foxp3+ CD4+ T cell response and that regulatory T cell induction at the site of inflammation precedes the effector T cell response. Induced Foxp3+ CD4+ T cells are highly suppressive ex vivo, demonstrating that influenza virus-induced Foxp3+ CD4+ T cells are phenotypically regulatory. Influenza A virus-induced regulatory T cells proliferate vigorously in response to influenza virus antigen, are disseminated throughout the site of infection and primary and secondary lymphoid organs, and retain Foxp3 expression in vitro, suggesting that acute viral infection is capable of inducing a foreign-antigen-specific Treg response. The ability of influenza virus-induced regulatory T cells to suppress antigen-specific CD4+ and CD8+ T cell proliferation and cytokine production correlates closely to their ability to respond to influenza virus antigens, suggesting that virus-induced Tregs are capable of attenuating effector responses in an antigen-dependent manner. Collectively, these data demonstrate that primary acute viral infection is capable of inducing a robust, antigen-responsive, and suppressive regulatory T cell response.

Rapid Detection of H5N1 Subtype Influenza Viruses by Antigen Capture Enzyme-Linked Immunosorbent Assay Using H5- and N1-Specific Monoclonal Antibodies

ABSTRACT Highly pathogenic avian influenza (HPAI) virus of the H5N1 subtype has caused devastating damage to poultry flocks and sporadic human H5N1 infections. There is concern that this virus subtype may gain transmissibility and become pandemic. Rapid diagnosis and surveillance for H5N1 subtype viruses are critical for the control of H5N1 infection. In this study, we report a robust antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) based on H5- and N1-specific monoclonal antibodies (MAbs) for the rapid detection of H5N1 subtype viruses. The H5 hemagglutinin (HA)-specific MAb (2D9) targets a conformational epitope which recognized multiple clades of H5N1 viruses, including clades 0, 1, 2.1, 2.2, 2.3, 4, 7, and 8. The N1 neuraminidase (NA)-specific MAb (8H12) recognized a linear epitope comprising the sequence AELPF. This epitope was 99% conserved in the NA of 708 analyzed H5N1 viruses, while the epitope was absent in NAs of subtypes N2 through N9. The specificity of the AC-ELISA was examined by using 41 H5N1 HPAI strains from multiple clades, 36 non-H5N1 viruses, and 4 influenza B viruses. No cross-reactivity was observed for any of the non-H5N1 viruses tested. The estimated detection limit was 1 to 2 HA titers. It is concluded that this H5N1 AC-ELISA can simultaneously detect H5 and N1 subtype antigens, eliminating the need for secondary testing for the NA subtype. Implementation of this assay in ELISA-like formats suitable for field use, such as dot ELISA, immunofiltration, or electrochemical biosensor technologies, would provide dual on-site detection of H5 and N1 in clinical or environmental specimens.

Reverse micelle-encapsulated recombinant baculovirus as an oral vaccine against H5N1 infection in mice

Induction of mucosal immunity through oral immunization is an effective way to control influenza infection. In this study, baculovirus displaying influenza hemagglutinin was encapsulated within a reverse micelle structure of phosphatidylcholine and delivered into the gastrointestinal tract of mice to study its efficacy as an oral vaccine against cross-clade H5N1 infection. Mice vaccinated with encapsulated baculovirus displaying HA (En-BacHA) showed significantly enhanced HA specific serum IgG and mucosal IgA antibodies, and higher hemagglutination inhibition (HI) titers, when compared to its non-encapsulated form (BacHA). Estimation of serum neutralizing antibodies also indicated that En-BacHA formulation was able to induce strong cross-clade neutralization against heterologous H5N1 strains (clade 1.0, clade 2.1, clade 4.0 and clade 8.0). Further, mice vaccinated with En-BacHA alone were able to confer 100% protection against 5MLD50 of HPAI heterologous H5N1 strain (clade 1). Inclusion of recombinant cholera toxin B subunit as a mucosal adjuvant in the vaccine formulation did not show any significant effect in both systemic and mucosal immune responses. Oral delivery of encapsulated recombinant H5 HA expressed on baculovirus surface is an effective way to prime the immune system against H5N1 infection in mice and will have no biosafety concerns associated with their production or administration.

Prophylactic and therapeutic efficacy of a chimeric monoclonal antibody specific for H5 haemagglutinin against lethal H5N1 influenza.

BACKGROUND Recent outbreaks of highly pathogenic H5N1 viruses in humans indicate that no endogenous protection exists in the general population. Vaccination programmes against this new pathogen require synthesis of endogenous antibodies and cannot provide any immediate protection in the event of a pandemic. Passive immunization with humanized neutralizing monoclonal antibodies can prove to be promising in preventing a catastrophic pandemic. METHODS A murine monoclonal antibody (mAb) 3B1 of immunoglobulin M isotype was switched to a chimeric immunoglobulin G1. BALB/c mice were used to study the protective efficacy of the chimeric mAbs against a lethal H5N1 virus challenge with strains from clades 1 and 2.1. Kinetics of the viral load were determined during the course of the treatment. RESULTS The chimeric mAb, in passive administration, was able to protect 100% of the mice when challenged with H5N1 strains from clades 1 or 2.1. Prophylaxis at 1 day prior to challenge and treatment at 1 day after challenge with this mAb resulted in the clearance of the virus from the lungs of the infected mice within 6 days post-viral challenge. CONCLUSIONS Passive immunotherapy using chimeric mAb 3B1 can be an effective tool in both the prophylaxis and treatment of highly pathogenic H5N1 infection, providing the immediate immunity needed to contain a future influenza pandemic.

Gamma Interferon Regulates Contraction of the Influenza Virus-Specific CD8 T Cell Response and Limits the Size of the Memory Population

ABSTRACT The factors that regulate the contraction of the CD8 T cell response and the magnitude of the memory cell population against localized mucosal infections such as influenza are important for generation of efficient vaccines but are currently undefined. In this study, we used a mouse model of influenza to demonstrate that the absence of gamma interferon (IFN-γ) or IFN-γ receptor 1 (IFN-γR1) leads to aberrant contraction of antigen-specific CD8 T cell responses. The increased accumulation of the effector CD8 T cell population was independent of viral load. Reduced contraction was associated with an increased fraction of CD8 T cells expressing the interleukin-7 receptor (IL-7R) at the peak of the response, resulting in enhanced numbers of memory/memory precursor cells in IFN-γ−/− and IFN-γR−/− compared to wild-type (WT) mice. Blockade of IL-7 within the lungs of IFN-γ−/− mice restored the contraction of influenza virus-specific CD8 T cells, indicating that IL-7R is important for survival and is not simply a consequence of the lack of IFN-γ signaling. Finally, enhanced CD8 T cell recall responses and accelerated viral clearance were observed in the IFN-γ−/− and IFN-γR−/− mice after rechallenge with a heterologous strain of influenza virus, confirming that higher frequencies of memory precursors are formed in the absence of IFN-γ signaling. In summary, we have identified IFN-γ as an important regulator of localized viral immunity that promotes the contraction of antigen-specific CD8 T cells and inhibits memory precursor formation, thereby limiting the size of the memory cell population after an influenza virus infection.