Contributions of the avian influenza virus HA, NA, and M2 surface proteins to the induction of neutralizing antibodies and protective immunity

Abstract

Highly pathogenic avian influenza virus (HPAIV) subtype H5N1 causes severe disease and mortality in poultry. Increased transmission of H5N1 HPAIV from birds to humans is a serious threat to public health. We evaluated the individual contributions of each of the three HPAIV surface proteins, namely, the hemagglutinin (HA), the neuraminidase (NA), and the M2 proteins, to the induction of HPAIV-neutralizing serum antibodies and protective immunity in chickens. Using reverse genetics, three recombinant Newcastle disease viruses (rNDVs) were engineered, each expressing the HA, NA, or M2 protein of H5N1 HPAIV. Chickens were immunized with NDVs expressing a single antigen (HA, NA, and M2), two antigens (HA+NA, HA+M2, and NA+M2), or three antigens (HA+NA+M2). Immunization with HA or NA induced high titers of HPAIV-neutralizing serum antibodies, with the response to HA being greater, thus identifying HA and NA as independent neutralization antigens. M2 did not induce a detectable neutralizing serum antibody response, and inclusion of M2 with HA or NA reduced the magnitude of the response. Immunization with HA alone or in combination with NA induced complete protection against HPAIV challenge. Immunization with NA alone or in combination with M2 did not prevent death following challenge, but extended the time period before death. Immunization with M2 alone had no effect on morbidity or mortality. Thus, there was no indication that M2 is immunogenic or protective. Furthermore, inclusion of NA in addition to HA in a vaccine preparation for chickens may not enhance the high level of protection provided by HA.

FIG. 1.

Construction and characterization of rNDVs expressing H5N1 HPAIV surface protein HA, NA, or M2. (A) Schematic diagram depicting the rNDV LaSota genome with insertion of an added gene engineered to express the HPAIV surface protein HA (construct 1), NA (construct 2), or M2 (construct 3). The inserted foreign ORF was preceded by an NDV gene junction containing gene-end (GE) and gene-start (GS) transcription signals. This placed the HA, NA, or M2 ORF under the control of a set of transcription signals such that each was expressed as a separate mRNA. (B) Expression of HPAIV surface proteins HA, NA, and M2 in DF1 cells and their incorporation into rNDV virions. DF1 cells were infected with the individual rNDV constructs, and 48 h later the cells were collected and processed to prepare cell lysates. In addition, allantoic fluid from embryonated eggs infected with the individual constructs was clarified and subjected to centrifugation on sucrose gradients to make partially purified preparations of virus particles. These samples were analyzed by Western blot analysis using a chicken antiserum generated by infection with H5N1 HPAIV (top) or a rabbit antiserum specific to a C-terminal peptide of the N1 NA protein (bottom). Lanes 1 to 4: lysates of DF1 cells that had been infected with rNDV empty vector (lane 1), rNDV-HA (lane 2), rNDV-NA (lane 3), or rNDV-M2 (lane 4). Lanes 5 to 8: partially purified virus particles of rNDV empty vector (lane 5), rNDV-HA (lane 6), rNDV-NA (lane 7), or rNDV-M2 (lane 8). The chicken HPAIV-specific antiserum detected the HA (lanes 2 and 5, top) and M2 (lanes 4 and 8, top) proteins but not the NA protein (lanes 3 and 7, top), whereas NA was detected with the NA-specific rabbit serum (lanes 3 and 7, bottom). The HA protein in cell lysates was detected as the uncleaved inactive precursor (HA0) and the proteolytically cleaved HA1 and HA2 proteins (lane 2, top), whereas only the last two species were detected in virus particles (lane 6, top). The rabbit NA-specific antibodies also detected nonspecific bands (NSP, bottom). nt, nucleotides; aa, amino acids.

FIG. 2.

Comparison of multicycle growth kinetics of rNDVs in embryonated chicken eggs (A) and in DF1 cells (B). (A) Nine-day-old embryonated chicken eggs were inoculated with 100 PFU of each virus, and allantoic fluids from three eggs were harvested at different time points (12 h, 24 h, 36 h, 48 h, 60 h, and 72 h) after inoculation. (B) DF1 cells were infected with 100 PFU (0.01 PFU per cell) of each virus, and the cell culture supernatant was harvested at 8-h intervals. The virus titer in allantoic fluid and cell culture supernatant samples was determined by TCID₅₀ assay in DF1 cells.

FIG. 3.

NDV- and HPAIV-specific serum antibody responses in chickens at 21 days following oculonasal immunization with the indicated rNDVs administered individually or in combination. NDV-specific serum antibody responses were determined by commercial NDV ELISA (A) and HI assay (B), whereas HPAIV-specific antibody responses were determined by AIV HI (C) and NAI assays (D). All antibody titers are expressed as mean reciprocal log₂ titer ± SEM (standard error of the mean), and the sample titers above the dotted line were considered positive. In the NDV ELISA (A), antibody titers were expressed as an S/P (sample/positive) ratio and titers above 0.3 (dotted line) were considered NDV positive. In the NDV (B) and HPAIV (C) 7 assays, titers were expressed as mean reciprocal log₂ titer, and titers above 1.0 log₂ (dotted line) were considered positive. In the HPAIV-specific NAI assay (D), antibody titers were expressed as the mean of the reciprocal log₂ titer at which a 50% reduction of NA activity was observed, and titers above 1.0 log₂ (dotted line) were considered positive. Statistical differences were calculated by one-way ANOVA with P < 0.001 in the NDV ELISA (A) and P < 0.0001 in the NDV HI (B), AIV HI (C), and AIV NAI (D) assays, respectively.

FIG. 4.

Western blot analysis of sera obtained from chickens immunized with rNDV-M2 individually or in combination with rNDV-HA or NDV-NA. Aliquots of partially purified recombinant AIV 6attWF10:2H5ΔN1, a recombinant AIV that was attenuated by removal of the polybasic cleavage site in HA, was subjected to electrophoresis in replicate lanes to serve as Western blot antigen. Western blot analysis was performed with postinfection HPAIV-specific chicken antiserum that detected the HA and M2 proteins (lane 1) or with a representative serum from a chicken immunized with rNDV-HA+M2 (lane 2), rNDV-NA+M2 (lane 3), or rNDV-HA+NA+M2 (lane 4) or with sera from individual chickens that had been immunized with rNDV-M2 (lanes 5 to 13).

FIG. 5.

Induction of HPAIV-neutralizing serum antibodies by rNDV vaccine constructs. Chickens were immunized once by the oculonasal route with the indicated rNDV constructs given individually or in combination, and sera were taken 21 days later and analyzed for the ability to neutralize the homologous A/Vietnam/1203/2004 strain in vitro. The serum-neutralizing antibody titers were expressed as mean reciprocal log₂ titer (means ± SEM), and titers above 1 log₂ (dotted line) were considered positive. Statistical differences were calculated by one-way ANOVA with P < 0.001.

FIG. 6.

Replication of HPAIV challenge virus in different organs of chickens infected 21 days after immunization with rNDVs. Three chickens from each immunized group were sacrificed 3 days after challenge. Various organs from each chicken were collected and analyzed in MDCK cells. The HPAIV titers in different organs of each chicken are expressed in log₁₀ TCID₅₀ per gram of tissue.

FIG. 7.

HPAIV challenge virus shedding titer as determined by TCID₅₀ assay in oral (A) and cloacal (B) swabs collected from chicken groups immunized with rNDVs. The virus titers are expressed as means ± SEM, and the sample titers above the dotted line (1.0 log₁₀) were considered positive. No virus was detected in the rNDV-HA and rNDV-HA+NA groups.

FIG. 8.

Percent survival of rNDV-immunized chickens following HPAIV challenge. Eight chicken groups (10 chickens per group) were immunized with the indicated rNDVs either individually (rNDV empty vector, rNDV-HA, rNDV-NA, and rNDV-M2) or in combination (rNDV-HA+NA, rNDV-HA+M2, rNDV-NA+M2, and rNDV-HA+NA+M2). Twenty-one days after immunization, these groups were challenged with H5N1 HPAIV. All of the chickens in the rNDV empty vector and rNDV-M2 groups died by day 2, all of the chickens in the rNDV-NA and rNDV-NA+M2 groups died by day 6, most of the animals in the rNDV-HA+M2 and rNDV-HA+NA+M2 groups survived, and all of the animals in the rNDV-HA and rNDV-HA+NA groups survived. Statistical differences between the groups were determined by the log-rank test with a P value of <0.0001.

FIG. 9.

Comparison of prechallenge (day 21) and postchallenge (day 31) HPAIV-specific serum antibody titers determined from surviving birds from the following immunization groups: rNDV-HA (n = 10), rNDV-HA+NA (n = 10), rNDV-HA+M2 (n = 8), and rNDV-HA+NA+M2 (n = 8). The HPAIV-specific serum antibody responses titers were measured by AIV NP ELISA (A), HI assay (B), and NAI assay (C). The AIV NP ELISA (A) titers were measured by S/P ratio, and values above 0.3 (dotted line) were considered positive. In the AIV HI (B) and NAI (C) assays, antibody titers were expressed as the mean reciprocal log₂ titer ± SEM. Titers above 1.0 log₂ were considered positive. The statistical difference between the chicken groups were analyzed by one-way ANOVA, and the P values were <0.001 for AIV NP ELISA and <0.0001 for the AIV HI and NAI assays.