The isolation of broadly neutralizing antibodies against influenza A viruses has been a long-sought goal for therapeutic approaches and vaccine design. Using a single-cell culture method for screening large numbers of human plasma cells, we isolated a neutralizing monoclonal antibody that recognized the hemagglutinin (HA) glycoprotein of all 16 subtypes and neutralized both group 1 and group 2 influenza A viruses. Passive transfer of this antibody conferred protection to mice and ferrets. Complexes with HAs from the group 1 H1 and the group 2 H3 subtypes analyzed by x-ray crystallography showed that the antibody bound to a conserved epitope in the F subdomain. This antibody may be used for passive protection and to inform vaccine design because of its broad specificity and neutralization potency.

https://science.sciencemag.org/content/sci/333/6044/850.full.pdf

A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins

Current flu vaccines provide only limited coverage against seasonal strains of influenza viruses. The identification of VH1-69 antibodies that broadly neutralize almost all influenza A group 1 viruses constituted a breakthrough in the influenza field. Here, we report the isolation and characterization of a human monoclonal antibody CR8020 with broad neutralizing activity against most group 2 viruses, including H3N2 and H7N7, which cause severe human infection. The crystal structure of Fab CR8020 with the 1968 pandemic H3 hemagglutinin (HA) reveals a highly conserved epitope in the HA stalk distinct from the epitope recognized by the VH1-69 group 1 antibodies. Thus, a cocktail of two antibodies may be sufficient to neutralize most influenza A subtypes and, hence, enable development of a universal flu vaccine and broad-spectrum antibody therapies.

/pdf/a-highly-conserved-neutralizing-epitope-on-group-2-influenza-3r3vd2za0j.pdf

A Highly Conserved Neutralizing Epitope on Group 2 Influenza A Viruses

Antibody VRC01 represents a human immunoglobulin that neutralizes -∼90% of diverse HIV-1 isolates. To understand how such broadly neutralizing HIV-1 antibodies develop and recognize the viral envelope, we used X-ray crystallography and 454 pyrosequencing to characterize additional antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding of different antibodies to the same CD4-binding-site epitope. Antibody recognition was achieved through the evolution of complementary contact domains that were generated in diverse ways. Phylogenetic analysis of expressed heavy and light chains determined by deep sequencing revealed a common pathway of antibody heavy chain maturation confined to IGHV1-2*02 lineage that could pair with different light chains. The maturation pathway inferred by antibodyomics reveals that diverse antibodies evolve to a highly affinity-matured state to recognize an invariant viral structure, providing insight into the development and evolution of broadly neutralizing HIV-1 immunity.

https://science.sciencemag.org/content/sci/333/6049/1593.full.pdf

Focused evolution of hiv-1 neutralizing antibodies revealed by crystal structures and deep sequencing

The role of T cells in mediating heterosubtypic protection against natural influenza illness in humans is uncertain. The 2009 H1N1 pandemic (pH1N1) provided a unique natural experiment to determine whether crossreactive cellular immunity limits symptomatic illness in antibody-naive individuals. We followed 342 healthy adults through the UK pandemic waves and correlated the responses of pre-existing T cells to the pH1N1 virus and conserved core protein epitopes with clinical outcomes after incident pH1N1 infection. Higher frequencies of pre-existing T cells to conserved CD8 epitopes were found in individuals who developed less severe illness, with total symptom score having the strongest inverse correlation with the frequency of interferon-γ (IFN-γ)(+) interleukin-2 (IL-2)(-) CD8(+) T cells (r = -0.6, P = 0.004). Within this functional CD8(+)IFN-γ(+)IL-2(-) population, cells with the CD45RA(+) chemokine (C-C) receptor 7 (CCR7)(-) phenotype inversely correlated with symptom score and had lung-homing and cytotoxic potential. In the absence of crossreactive neutralizing antibodies, CD8(+) T cells specific to conserved viral epitopes correlated with crossprotection against symptomatic influenza. This protective immune correlate could guide universal influenza vaccine development.

Cellular immune correlates of protection against symptomatic pandemic influenza

Identification of broadly neutralizing antibodies against influenza A viruses has raised hopes for the development of monoclonal antibody-based immunotherapy and "universal" vaccines for influenza. However, a substantial part of the annual flu burden is caused by two cocirculating, antigenically distinct lineages of influenza B viruses. Here, we report human monoclonal antibodies, CR8033, CR8071, and CR9114, that protect mice against lethal challenge from both lineages. Antibodies CR8033 and CR8071 recognize distinct conserved epitopes in the head region of the influenza B hemagglutinin (HA), whereas CR9114 binds a conserved epitope in the HA stem and protects against lethal challenge with influenza A and B viruses. These antibodies may inform on development of monoclonal antibody-based treatments and a universal flu vaccine for all influenza A and B viruses.

/pdf/highly-conserved-protective-epitopes-on-influenza-b-viruses-2boha3auuy.pdf

Highly Conserved Protective Epitopes on Influenza B Viruses

The 2009 pandemic H1N1 influenza pandemic demonstrated the global health threat of reassortant influenza strains. Herein, we report a detailed analysis of plasmablast and monoclonal antibody responses induced by pandemic H1N1 infection in humans. Unlike antibodies elicited by annual influenza vaccinations, most neutralizing antibodies induced by pandemic H1N1 infection were broadly cross-reactive against epitopes in the hemagglutinin (HA) stalk and head domain of multiple influenza strains. The antibodies were from cells that had undergone extensive affinity maturation. Based on these observations, we postulate that the plasmablasts producing these broadly neutralizing antibodies were predominantly derived from activated memory B cells specific for epitopes conserved in several influenza strains. Consequently, most neutralizing antibodies were broadly reactive against divergent H1N1 and H5N1 influenza strains. This suggests that a pan-influenza vaccine may be possible, given the right immunogen. Antibodies generated potently protected and rescued mice from lethal challenge with pandemic H1N1 or antigenically distinct influenza strains, making them excellent therapeutic candidates.

/pdf/broadly-cross-reactive-antibodies-dominate-the-human-b-cell-575p74gdn7.pdf

Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection

Purpose Blockade of the CD40/CD154 T cell costimulation pathway is a promising approach to replace current clinical immunosuppression. TNX-1500 (TNX) is a novel humanized anti-CD154 mAb that contains the hu5c8 Fab region and an Fc region engineered to modulate FcγR2 binding to reduce the risk of pro-thrombotic platelet activation and aggregation, and thus potentially prevent the thromboembolic events seen with hu5c8 IgG1 in preclinical models and clinical trials. Methods Cynomolgus monkey heterotopic cardiac allograft recipients were treated with TNX either using a ‘standard’ regimen (sTNX; 30 mg/kg iv twice weekly x4, then 20 mg/kg weekly from d21, n=5), using a reduced-dose TNX maintenance regimen (loTNX; 30 mg/kg iv twice weekly x4, 10 mg/kg weekly x6, then 20 mg/kg monthly; n=4), or loTNX with mycophenolate mofetil (loTNX+MMF, 200mg/d po, n=4); results were compared to historical data using hu5c8 (n=5). Graft function was monitored by telemetry and ultrasound until graft failure or elective experimental termination at 6 months. Protocol biopsy and explanted graft histology and anti-donor antibody (alloAb) were evaluated. Results Four of five grafts treated with sTNX maintained good graft function (MST >154d, range 114-180, 4 of 5 ongoing); one graft rejected at 114d. All grafts treated with loTNX, alone or with MMF, experienced acute cellular rejection and/or antibody-mediated rejection; 7 of 8 grafts failed before 6 months. Relative to hu5c8 alone (MST 123d), loTNX (MST 99d) and sTNX (MST>154d) showed a statistically similar prolongation of cardiac allograft survival. Although loTNX+MMF attenuated IgG alloAb production during treatment (4/4 animals) more consistently than loTNX alone (2/4), cardiac allograft vasculopathy (CAV) tended to be more prominent with loTNX+MMF; alloAb and CAV in the sTNX group after study completion will be presented. Neither sustained thrombocytopenia nor micro- or macrovascular thrombosis were observed in TNX-treated animals. Conclusion Blockade of CD154 with TNX monotherapy consistently and safely prevents pathologic alloimmunity in this stringent preclinical model, similar to hu5c8 monotherapy. Lower maintenance dosing with TNX, with or without MMF, was less effective. Blockade of the CD40/CD154 T cell costimulation pathway is a promising approach to replace current clinical immunosuppression. TNX-1500 (TNX) is a novel humanized anti-CD154 mAb that contains the hu5c8 Fab region and an Fc region engineered to modulate FcγR2 binding to reduce the risk of pro-thrombotic platelet activation and aggregation, and thus potentially prevent the thromboembolic events seen with hu5c8 IgG1 in preclinical models and clinical trials. Cynomolgus monkey heterotopic cardiac allograft recipients were treated with TNX either using a ‘standard’ regimen (sTNX; 30 mg/kg iv twice weekly x4, then 20 mg/kg weekly from d21, n=5), using a reduced-dose TNX maintenance regimen (loTNX; 30 mg/kg iv twice weekly x4, 10 mg/kg weekly x6, then 20 mg/kg monthly; n=4), or loTNX with mycophenolate mofetil (loTNX+MMF, 200mg/d po, n=4); results were compared to historical data using hu5c8 (n=5). Graft function was monitored by telemetry and ultrasound until graft failure or elective experimental termination at 6 months. Protocol biopsy and explanted graft histology and anti-donor antibody (alloAb) were evaluated. Four of five grafts treated with sTNX maintained good graft function (MST >154d, range 114-180, 4 of 5 ongoing); one graft rejected at 114d. All grafts treated with loTNX, alone or with MMF, experienced acute cellular rejection and/or antibody-mediated rejection; 7 of 8 grafts failed before 6 months. Relative to hu5c8 alone (MST 123d), loTNX (MST 99d) and sTNX (MST>154d) showed a statistically similar prolongation of cardiac allograft survival. Although loTNX+MMF attenuated IgG alloAb production during treatment (4/4 animals) more consistently than loTNX alone (2/4), cardiac allograft vasculopathy (CAV) tended to be more prominent with loTNX+MMF; alloAb and CAV in the sTNX group after study completion will be presented. Neither sustained thrombocytopenia nor micro- or macrovascular thrombosis were observed in TNX-treated animals. Blockade of CD154 with TNX monotherapy consistently and safely prevents pathologic alloimmunity in this stringent preclinical model, similar to hu5c8 monotherapy. Lower maintenance dosing with TNX, with or without MMF, was less effective. 

Zahida Ali

Papers

Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection

TNX-1500, an Fc-modified Anti-CD154 Antibody, Prolongs Nonhuman Primate Renal Allograft Survival.