measuring-influenza-neutralizing-antibody-responses-to-h3n2-viruses

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Centers for Disease Control and Prevention

Swine origin 2009 H1N1 influenza virus has spread globally to cause the first influenza pandemic of the 21st century. Serological studies can improve our understanding of the extent of human infection and risk factors associated with the transmission of this pandemic virus. The "gold standard" for serodiagnosis of human influenza virus infection is the detection of seroconversion between acute- and convalescent-stage samples. However, the timing of seroepidemiological investigations often precludes the collection of truly acute-phase sera, requiring development of serological criteria for evaluating convalescent-phase sera that optimize detection of true positives and true negatives. To guide seroepidemiological investigations into the spread of the novel 2009 pandemic H1N1 virus, we characterized serum antibody responses to 2009 H1N1 virus in 87 individuals with confirmed viral infection and 227 nonexposed U.S. individuals using microneutralization (MN) and hemagglutination inhibition (HI) assays. Sensitivity and specificity were determined for each assay alone and in combination for detection of 2009 H1N1 virus-specific antibodies in convalescent-phase sera. Although the HI assay was more specific for detecting antibody to 2009 H1N1, the MN assay was more sensitive, particularly for detecting low-titer seroconversions. A combination of titers (MN ≥ 40 and HI ≥ 20) provided the highest sensitivity (90%) and specificity (96%) for individuals aged <60 years and 92% specificity for adults aged ≥ 60 years for detection of serologically confirmed 2009 H1N1 infections in U.S. populations during the first pandemic waves. These studies provide an approach to optimize timely serological investigations for future pandemics or outbreaks of novel influenza viruses among humans.

Sensitivity and specificity of serologic assays for detection of human infection with 2009 pandemic H1N1 virus in U.S. populations.

The first two laboratory-confirmed cases of 2009 pandemic influenza A (H1N1) virus (H1N1pdm09) infection were detected in San Diego (SD) and Imperial County (IC) in southern California, April 2009.

The first cases of 2009 pandemic influenza A (H1N1) virus infection in the United States: a serologic investigation demonstrating early transmission.

The recommendation by the American Society of Transplantation for annual trivalent inactivated influenza vaccination greater than 3 to 6 months post-kidney transplantation provides a unique opportunity to test the in vivo impact of immunosuppression on recall T- and B-cell responses to influenza vaccination.

Impact of immunosuppression on recall immune responses to influenza vaccination in stable renal transplant recipients.

Background.  Influenza disproportionately impacts older adults while current vaccines have reduced effectiveness in the older population. Methods.  We conducted a comprehensive evaluation of cellular and humoral immune responses of adults aged 50 years and older to the 2008-2009 seasonal trivalent inactivated influenza vaccine and assessed factors influencing vaccine response. Results.  Vaccination increased hemagglutination inhibition and neutralizing antibody; however, 66.3% of subjects did not reach hemagglutination inhibition titers ≥ 40 for H1N1, compared with 22.5% for H3N2. Increasing age had a minor negative impact on antibody responses, whereas prevaccination titers were the best predictors of postvaccination antibody levels. Preexisting memory B cells declined with age, especially for H3N2. However, older adults still demonstrated a significant increase in antigen-specific IgG(+) and IgA(+) memory B cells postvaccination. Despite reduced frequency of preexisting memory B cells associated with advanced age, fold-rise in memory B cell frequency in subjects 60+ was comparable to subjects age 50-59. Conclusions.  Older adults mounted statistically significant humoral and cell-mediated immune responses, but many failed to reach hemagglutination inhibition titers ≥40, especially for H1N1. Although age had a modest negative effect on vaccine responses, prevaccination titers were the best predictor of postvaccination antibody levels, irrespective of age.

Preexisting Immunity, More Than Aging, Influences Influenza Vaccine Responses.

It is well established that virus neutralizing (VN) antibodies to hemagglutinin (HA) antigens of influenza A viruses provide optimal protection against antigenically matched strains of influenza A viruses. In contrast, little is known about the potential role of HA-specific, non-neutralizing antibodies in protection against human influenza illness at present. In this study, we show that individuals vaccinated with the 2014-15 seasonal inactivated influenza vaccine displayed strong A/H3N2 HA-specific antibody-dependent cell-mediated cytotoxicity (ADCC) activities against an antigenically drifted H3N2 virus, despite poor induction of cross-reactive neutralizing antibodies against the antigenic variant. Given that passive transfer of influenza HA-monospecific immune sera with negligible levels of HA-specific VN antibodies can often confer considerable cross protection against lethal challenge with heterologous influenza viruses in animal models, it is conceivable that HA-specific, non-neutralizing antibodies may provide certain degree of cross protection against antigenically drifted influenza A viruses through ADCC in case of influenza vaccine mismatches. This may have important implications for public health.

Vaccination with 2014-15 Seasonal Inactivated Influenza Vaccine Elicits Cross-Reactive Anti-HA Antibodies with Strong ADCC Against Antigenically Drifted Circulating H3N2 Virus in Humans.

Background.  Detection of neutralizing antibodies (nAbs) to influenza A virus hemagglutinin (HA) antigens by conventional serological assays is currently the main immune correlate of protection for influenza vaccines However, current prepandemic avian influenza vaccines are poorly immunogenic in inducing nAbs despite considerable protection conferred. Recent studies show that Ab-dependent cell-mediated cytotoxicity (ADCC) to HA antigens are readily detectable in the sera of healthy individuals and patients with influenza infection. Methods.  Virus neutralization and ADCC activities of serum samples from individuals who received either seasonal or a stock-piled H5N1 avian influenza vaccine were evaluated by hemagglutination inhibition assay, microneutralization assay, and an improved ADCC natural killer (NK) cell activation assay. Results.  Immunization with inactivated seasonal influenza vaccine led to strong expansion of both nAbs and ADCC-mediating antibodies (adccAbs) to H3 antigen of the vaccine virus in 24 postvaccination human sera. In sharp contrast, 18 individuals vaccinated with the adjuvanted H5N1 avian influenza vaccine mounted H5-specific antibodies with strong ADCC activities despite moderate virus neutralization capacity. Strength of HA-specific ADCC activities is largely associated with the titers of HA-binding antibodies and not with the fine antigenic specificity of anti-HA nAbs. Conclusions.  Detection of both nAbs and adccAbs may better reflect protective capacity of HA-specific antibodies induced by avian influenza vaccines.

Antibody-Dependent Cell-Mediated Cytotoxicity to Hemagglutinin of Influenza A Viruses After Influenza Vaccination in Humans.

Zoonotic A(H7N9) avian influenza viruses emerged in China in 2013 and continue to be a threat to human public health, having infected over 800 individuals with a mortality rate approaching 40%. Treatment options for people infected with A(H7N9) include the use of neuraminidase (NA) inhibitors. However, like other influenza viruses, A(H7N9) can become resistant to these drugs. The use of monoclonal antibodies is a rapidly developing strategy for controlling influenza virus infection. Here we generated a murine monoclonal antibody (3c10-3) directed against the NA of A(H7N9) and show that prophylactic systemic administration of 3c10-3 fully protected mice from lethal challenge with wild-type A/Anhui/1/2013 (H7N9). Further, post-infection treatment with a single systemic dose of 3c10-3 at either 24, 48 or 72 h post A(H7N9) challenge resulted in both dose- and time-dependent protection of up to 100% of mice, demonstrating therapeutic potential for 3c10-3. Epitope mapping revealed that 3c10-3 binds near the enzyme active site of NA, and functional characterization showed that 3c10-3 inhibits the enzyme activity of NA and restricts the cell-to-cell spread of the virus in cultured cells. Affinity analysis also revealed that 3c10-3 binds equally well to recombinant NA of wild-type A/Anhui/1/2013 and to a variant NA carrying a R289K mutation known to infer NAI resistance. These results suggest that 3c10-3 has the potential to be used as a therapeutic to treat A(H7N9) infections either as an alternative to, or in combination with, current NA antiviral inhibitors.

An influenza A virus (H7N9) anti-neuraminidase monoclonal antibody with prophylactic and therapeutic activity in vivo.

Avian influenza A (H5N1) viruses represent a growing threat for an influenza pandemic. The presence of widespread avian influenza virus infections further emphasizes the need for vaccine strategies for control of pre-pandemic H5N1 and other avian influenza subtypes. Influenza neuraminidase (NA) vaccines represent a potential strategy for improving vaccines against avian influenza H5N1 viruses. To evaluate a strategy for NA vaccination, we generated a recombinant influenza virus-like particle (VLP) vaccine comprised of the NA protein of A/Indonesia/05/2005 (H5N1) virus. Ferrets vaccinated with influenza N1 NA VLPs elicited high-titer serum NA-inhibition (NI) antibody titers and were protected from lethal challenge with A/Indonesia/05/2005 virus. Moreover, N1-immune ferrets shed less infectious virus than similarly challenged control animals. In contrast, ferrets administered control N2 NA VLPs were not protected against H5N1 virus challenge. These results provide support for continued development of NA-based vaccines against influenza H5N1 viruses.

Neuraminidase-based recombinant virus-like particles protect against lethal avian influenza A(H5N1) virus infection in ferrets.

The emergence of A(H7N9) virus strains with resistance to neuraminidase (NA) inhibitors highlights a critical need to discover new countermeasures for treatment of A(H7N9) virus-infected patients. We previously described an anti-NA mAb (3c10-3) that has prophylactic and therapeutic efficacy in mice lethally challenged with A(H7N9) virus when delivered intraperitoneally (i.p.). Here we show that intrananasal (i.n.) administration of 3c10-3 protects 100% of mice from mortality when treated 24h post-challenge and further characterize the protective efficacy of 3c10-3 using a nonlethal A(H7N9) challenge model. Administration of 3c10-3 i.p. 24h prior to challenge resulted in a significant decrease in viral lung titers and deep sequencing analysis indicated that treatment did not consistently select for viral variants in NA. Furthermore, prophylactic administration of 3c10-3 did not inhibit the development of protective immunity to subsequent homologous virus re-challenge. Taken together, 3c10-3 highlights the potential use of anti-NA mAb to mitigate influenza virus infection.

An influenza A virus (H7N9) anti-neuraminidase monoclonal antibody protects mice from morbidity without interfering with the development of protective immunity to subsequent homologous challenge.

Article	Year
Sensitivity and specificity of serologic assays for detection of human infection with 2009 pandemic H1N1 virus in U.S. populations. Journal of clinical microbiology\| PubMed ID: 21471339	2011
The first cases of 2009 pandemic influenza A (H1N1) virus infection in the United States: a serologic investigation demonstrating early transmission. Influenza and other respiratory viruses\| PubMed ID: 22353441	2012
Impact of immunosuppression on recall immune responses to influenza vaccination in stable renal transplant recipients. Transplantation\| PubMed ID: 24366008	2014
Preexisting Immunity, More Than Aging, Influences Influenza Vaccine Responses. Open forum infectious diseases\| PubMed ID: 26380344	2015
Vaccination with 2014-15 Seasonal Inactivated Influenza Vaccine Elicits Cross-Reactive Anti-HA Antibodies with Strong ADCC Against Antigenically Drifted Circulating H3N2 Virus in Humans. Viral immunology\| PubMed ID: 26950058	2016
Antibody-Dependent Cell-Mediated Cytotoxicity to Hemagglutinin of Influenza A Viruses After Influenza Vaccination in Humans. Open forum infectious diseases\| PubMed ID: 27419174	2016
An influenza A virus (H7N9) anti-neuraminidase monoclonal antibody with prophylactic and therapeutic activity in vivo. Antiviral research\| PubMed ID: 27713074	2016
Neuraminidase-based recombinant virus-like particles protect against lethal avian influenza A(H5N1) virus infection in ferrets. Virology\| PubMed ID: 28624679	2017
An influenza A virus (H7N9) anti-neuraminidase monoclonal antibody protects mice from morbidity without interfering with the development of protective immunity to subsequent homologous chall... Virology\| PubMed ID: 28888111	2017

Yaohui Bai

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