Conceived and designed the experiments: PZ. Performed the experiments: HD CGT FZ PB VD. Analyzed the data: HD CGT VD PZ. Contributed reagents/materials/analysis tools: FZ. Wrote the paper: PZ.
The authors have declared that no competing interests exist.
The spread of highly pathogenic avian influenza (HPAI) H5N1 virus in human remains a global health concern. Heterosubtypic antibody response between seasonal influenza vaccine and potential pandemic influenza virus has important implications for public health. Previous studies by Corti
In this study, using a sensitive influenza HA (hemagglutinin) and NA (neuraminidase) pseudotype-based neutralization (PN) assay we first confirmed that low levels of heterosubtypic neutralizing antibody response against H5N1 virus were indeed elicited with seasonal influenza vaccine in humans. We then immunized mice with the seasonal influenza vaccine and challenged them with lethal doses of highly pathogenic H5N1 virus. As controls, we immunized mice with homosubtypic H5N1 virus like particles (VLP) or PBS and challenged them with the same H5N1 virus. Here we show that low levels of heterosubtypic neutralizing antibody response were elicited with seasonal influenza vaccine in mice, which were significantly higher than those in PBS control. Among them 2 out of 27 whose immune sera exhibited similar levels of neutralizing antibody response as VLP controls actually survived from highly pathogenic H5N1 virus challenge.
Therefore, we conclude that low levels of heterosubtypic neutralizing antibody response are indeed elicited with seasonal influenza vaccine in humans and mice and at certain levels such response offers immune protection against severity of H5N1 virus infection.
Influenza A viruses are segmented, negative-strand RNA viruses in the family
Neutralizing antibody responses play an important role in prevention and clearance of influenza A virus infection. However, neutralizing antibodies are completely protective against secondary infections only with closely related influenza A strains, but ineffective against viruses with major antigenic divergence. Because of this, current seasonal influenza vaccines, which consists of human influenza A viruses of H1N1 and H3N2 subtypes and an influenza B virus, are prepared annually on the basis of WHO forecasts on the most probable influenza A and B virus strains thought to be circulating in the next seasonal outbreak
Previously, Gioia
Previously, we developed a sensitive influenza HA and NA pseudotype-based neutralization (PN) assay
Thus, in this study, using the sensitive PN assay we first tested if antibody responses elicited with seasonal influenza vaccines in healthy individuals can cross-neutralize H5N1 viruses. We hypothesized that heterosubtypic neutralizing antibody response against H5N1 viruses elicited with seasonal influenza vaccines may be too low to be consistently detected by conventional HI and MN assays, but can be readily detected by the sensitive PN assay. To accomplish this, 12 healthy volunteers were recruited and vaccinated with 2008–2009 seasonal influenza vaccines. Neutralizing antibody responses in the pre- and post-immune serum samples were evaluated by two modified PN assays - PN entry assay and PN release/entry assay - against a panel of HA and NA pseudotypes derived from H1N1, H3N2 and H5N1 viruses. We then immunized mice with the seasonal influenza vaccine, homosubtypic H5N1 VLP or PBS control and challenged them with lethal doses of highly pathogenic H5N1 virus. Neutralizing antibody responses against H1N1, H3N2 and H5N1 viruses in pre- and post-immune sera were evaluated using PN entry assay. Here we show that low levels of heterosubtypic neutralizing antibody response were indeed elicited with seasonal influenza vaccine in humans and in mice. Among them 2 out of 27 mice whose immune sera exhibited similar levels of neutralizing antibody response as VLP controls survived from highly pathogenic H5N1 virus challenge. Therefore, we conclude that low levels of heterosubtypic neutralizing antibody response can indeed be elicited with seasonal influenza vaccine in humans and mice and such response, when reached at certain levels, offers immune protection against severity of H5N1 virus infection.
Our human study was approved by the research and ethics committee in the Institut Pasteur of Shanghai. All human participants gave written consent. Our animal study was approved by animal research committee in Pasteur Institute in Cambodia and was carried out according to international guideline of animal research.
Twelve healthy young adult volunteers who were going to receiving 2008–2009 seasonal influenza vaccine gave written consent for sera to be taken prior to and after the vaccination at the Institut Pasteur of Shanghai, Chinese Academy of Sciences. The study was approved by the Institution Ethic Committee. The vaccine formulation was Vaxigrip, an inactivated split influenza vaccine (Sanofi Pasteur, Lyon, France). The antigen composition and strains were A/Brisbane/59/2007 (H1N1), A/Brisbane/10/2007 (H3N2) and B/Florida/4/2006 like. Each 0.5 ml vaccine dose contains 15 µg HA of each strain in PBS and excipients. Vaccine was administered intramuscularly. Blood samples were drawn 1 or 2 months before and 10 days after the vaccination. After clotting at room temperature for 6 hours and then at 4°C overnight, serum samples were collected, heat-inactivated at 56°C for 30 minutes and stored in aliquot at −80°C.
The packaging cell line 293T
HPAI H5N1 viruses A/Shenzhen/406H/06 and A/Cambodia/P0322095/05 were originally isolated from H5N1 infected human patients at the Donghu Hospital in Shenzhen, China
HA and NA pseudotype panel used in this study was generated as described previously
To test effect of pre- and post-immune sera on the entry of HA and NA pseudotypes, MDCK cells (2×104 cells per well) were seeded onto 24 well culture plate in complete DMEM overnight. Serially 4-fold diluted human sera or serially 2-fold diluted mouse sera (starting at 1∶10 dilution) were then incubated with various pseudotypes equivalent to 100,000–500,000 relative luciferase activity (RLA) at the final volume of 50 µl at 37°C for 1 hour. The mixture was added onto MDCK cells and incubated for 2 days. RLA in the cell lysates was measured as described before
To test the effect of pre- and post-immune sera on HA and NA pseudotype release, 4×105 293 T packaging cells were co-transfected with 1.4 µg transfer vector pHR'CMV-Luc
MDCK cells (1.5×104 cells per well) were seeded onto 48 well culture plate in complete DMEM overnight. To test neutralization activity of human pre- and post-immune serum samples, serially 4-fold diluted sera (starting at 1∶10 dilution) were incubated with 100 TCID50 wild type viruses A/Shenzhen/406H/06, A/Cambodia/P0322095/05 and A/WSN/33 at the final volume of 50 µl at 37°C for 1 hour. After the incubation, the mixture was added onto MDCK cells. The cytopathic effect (CPE) was scored at 4 days after infection as described before
All animal protocols were approved by the Institutional Animal Care and Use Committee at the Institut Pasteur in Cambodia (Permit number VD100820). Female BALB/c mice (Mus musculus) at the age of 6 to 8 weeks were purchased from Charles River Laboratories (L'Arbresle, France) and housed in micro-isolator cages ventilated under negative pressure with HEPA-filtered air and a 12/12-hour light/dark cycle.
The production and characterization of influenza HA and NA VLP were the same as described before
To characterize VLP, resuspended pellets were fractionated through a 25–65% sucrose density gradient at 25,000 rpm for 16 hours at 4°C in a Beckman SW41 swing rotor (Beckman Coulter, Fullerton, CA). Twelve fractions (0.96 ml each) were collected from the top to the bottom of the gradient, TCA precipitated, separated by 12% SDS-PAGE and transferred onto PDVF membranes. Blots were blocked in a solution of Tris-buffered saline containing 5% nonfat dry milk and 0.1% Tween 20 and subsequently probed with a monoclonal antibody (clone 183-H12) specific for HIV-1 gag p24 (provided by the AIDS Reagents and Depositary program, NIAID, NIH), with a monoclonal antibody (Catalog# F3165, Sigma) specific for M2 epitope and with immune sera elicited with DNA plasmids expressing H5HA A/Shenzhen/406H/06) (see below). Antigens were visualized with an AP-conjugated anti-mouse IgG antibody according to manufacturer's instruction (Promega).
Two immunization and challenge experiments were performed. In the first experiment, female BALB/c mice at the age of 6 to 8 weeks were randomly divided into 3 groups (5 mice per group). Mice in group one were injected intramuscularly (i.m.) with total 200 µl PBS (pH 7.4) in both prime and boost. Mice in group two were injected i.m. with 0.5 µg (based on HA content) HA and NA VLP in total 200 µl PBS for both prime and boost. Mice in group three were injected i.m. with the same 2008–2009 seasonal influenza vaccine as used in humans (see above). Each 200 µl vaccine dose contains total 1 µg HA in PBS and excipients. The prime and the boost were carried 21 days apart. Seven days before the prime and 7 days after the boost serum samples were collected, heat-inactivated at 56°C and stored in aliquot at −80°C. Two weeks after the boost, mice in each group were challenged i.n. with 10 MLD50 of H5N1 virus (A/Shenzhen/406H/06, subclade 2.3.4) in a volume of 50 µl. Mice were monitored and recorded daily for 14 days post challenge. When mice lose 25% or more of their initial body weight, they were sacrificed and counted as dead mice. Virus challenge studies were conducted in BSL-3 facility at the Institut Pasteur in Cambodia in accordance with the Department of Agriculture guidelines for the Care and Use of Laboratory Animals, the Animal Welfare Act and Department of Agriculture Biosafety guidelines in Microbiological and Biomedical Laboratory.
Since in the first immunization and challenge experiment all 5 PBS control mice died, all 5 VLP control mice survived and one of 5 seasonal influenza vaccinated mice whose immune serum reached similar level as VLP controls actually survived from lethal H5N1 challenge, we carried out the second immunization and challenge experiment. In the second experiment 2 mice were injected with PBS, 2 mice with VLP and 22 mice with seasonal influenza vaccine followed by the same H5N1 challenge.
Each individual animal immune response was counted as an individual value for statistical analysis. The neutralizing activity was first analyzed by the Kolmogorov-Smirnov and Shapiro-Wilk normality tests for normality and then the significance was calculated by Student's
H1N1 | Vaccine antigen strain | |||||||
A/WSN/33 | H1N1 A/Brisbane/59/2007 | H3N2 A/Brisbane/10/2007 | VSV | |||||
Human projects | IC50 | IC90 | IC50 | IC90 | IC50 | IC90 | IC50 | |
Pre- | 1 | <1∶10 |
<1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 |
immune | 2 | 1∶10–1∶40 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 |
sera | 3 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 |
4 | 1∶40–1∶160 | 1∶10–1∶40 | 1∶10–1∶40 | <1∶10 | 1∶10–1∶40 | <1∶10 | <1∶10 | |
5 | 1∶160–1∶640 | 1∶10–1∶40 | 1∶40 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | |
6 | 1∶160–1∶640 | 1∶40 | 1∶10 | <1∶10 | 1∶10 | <1∶10 | <1∶10 | |
7 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | |
8 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | |
9 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | |
10 | 1∶160–1∶640 | 1∶40 | 1∶10 | <1∶10 | 1∶10 | <1∶10 | <1∶10 | |
11 | 1∶40–1∶160 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | |
12 | 1∶160–1∶640 | 1∶40 | 1∶40 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | |
Post- | 1 | >1∶2560 |
1∶640–1∶2560 | >1∶2560 | 1∶640–1∶2560 | 1∶640–1∶2560 | 1∶40 | <1∶10 |
Immune | 2 | 1∶640 | 1∶40 | 1∶640–1∶2560 | 1∶40–1∶160 | 1∶160–1∶640 | 1∶10–1∶40 | <1∶10 |
sera | 3 | 1∶640 | 1∶40–1∶160 | 1∶640–1∶2560 | 1∶40–1∶160 | 1∶160–1∶640 | 1∶10–1∶40 | <1∶10 |
4 | >1∶2560 | 1∶40–1∶160 | >1∶2560 | 1∶160–1∶640 | 1∶160–1∶640 | 1∶40 | <1∶10 | |
5 | 1∶640–1∶2560 | 1∶160–1∶640 | 1∶640–1∶2560 | 1∶160–1∶640 | 1∶40–1∶160 | 1∶10 | <1∶10 | |
6 | 1∶640–1∶2560 | 1∶40–1∶160 | 1∶640–1∶2560 | 1∶160–1∶640 | 1∶40–1∶160 | 1∶10 | <1∶10 | |
7 | >1∶2560 | 1∶640–1∶2560 | >1∶2560 | 1∶640–1∶2560 | 1∶160–1∶640 | 1∶10–1∶40 | <1∶10 | |
8 | 1∶640 | 1∶40 | 1∶640–1∶2560 | 1∶40–1∶160 | 1∶40–1∶160 | <1∶10 | <1∶10 | |
9 | 1∶640–1∶2560 | 1∶160–1∶640 | >1∶2560 | 1∶160–1∶640 | 1∶40–1∶160 | 1∶10–1∶40 | <1∶10 | |
10 | 1∶640–1∶2560 | 1∶160–1∶640 | >1∶2560 | 1∶160–1∶640 | 1∶40–1∶160 | 1∶10 | <1∶10 | |
11 | >1∶2560 | 1∶640–1∶2560 | >1∶2560 | 1∶640–1∶2560 | 1∶160–1∶640 | 1∶40 | <1∶10 | |
12 | 1∶640–1∶2560 | 1∶160–1∶640 | 1∶640–1∶2560 | 1∶160–1∶640 | 1∶40–1∶160 | 1∶10–1∶40 | <1∶10 |
*<1∶10: not detected.
**>1∶2560: the Ab titers greater than the highest antibody dilutions.
H5N1 | |||||||
A/Hongkong/156/97 clade 0 | A/Vietnam/1203/04 clade 1 | A/Cambodia/P0322095/05 clade 1 | A/Indonesia/5/05 clade 2.1 | A/Shenzhen/406H/06 clade 2.3.4 | A/Silky Chicken/Hongkong/SF189/01 clade 3 | ||
Human projects | IC50 | IC50 | IC50 | IC50 | IC50 | IC50 | |
Pre- | 1 | <1∶10 |
<1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 |
immune | 2 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 |
sera | 3 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 |
4 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | |
5 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | |
6 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | |
7 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | |
8 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | |
9 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | |
10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | |
11 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | |
12 | <1∶10 | 1∶10–1∶40 | <1∶10 | <1∶10 | 1∶10 | <1∶10 | |
Post- | 1 | 1∶40 | 1∶160 | 1∶640 | 1∶640 | 1∶160–1∶640 | 1∶10 |
immune | 2 | 1∶40–1∶160 | 1∶40 | 1∶160 | 1∶160 | 1∶40 | 1∶40 |
sera | 3 | 1∶10 | <1∶10 | <1∶10 | 1∶40 | 1∶10 | <1∶10 |
4 | 1∶160–1∶640 | 1∶160 | 1∶40 | 1∶160 | 1∶160 | 1∶10–1∶40 | |
5 | <1∶10 | 1∶160 | 1∶40–1∶160 | 1∶10–1∶40 | <1∶10 | 1∶160 | |
6 | 1∶40 | 1∶160 | <1∶10 | 1∶160 | <1∶10 | 1∶40–1∶160 | |
7 | 1∶160 | 1∶10–1∶40 | 1∶160 | 1∶40 | <1∶10 | 1∶10 | |
8 | 1∶40 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | |
9 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | |
10 | <1∶10 | <1∶10 | <1∶10 | <1∶10 | 1∶40 | <1∶10 | |
11 | 1∶10–1∶40 | 1∶40 | <1∶10 | 1∶10 | 1∶10 | <1∶10 | |
12 | 1∶10–1∶40 | 1∶40–1∶160 | <1∶10 | <1∶10 | 1∶40 | <1∶10 |
*<1∶10: not detected.
Before 2008–2009 seasonal influenza vaccination, sera from 7, 5 or 3 of 12 donors exhibited low, but measurable neutralization titers (IC50 ranging from 1∶10 to 1∶320) against H1N1 A/WSN/33 and A/Brisbane/59/2007 or H3N2 A/Brisbane/10/2007 pseudotypes (
After 2008–2009 seasonal influenza vaccination, sera from all 12 donors exhibited much higher neutralization titers (IC50 ranging from 1∶640 to >2560 and IC90 ranging from 1∶40 to 1∶2,560) against H1N1 A/WSN/33 and A/Brisbane/59/2007 or H3N2 A/Brisbane/10/2007 pseudotypes, but none of them against pseudotype expressing VSV-G controls. Interestingly, post-immune sera from 11 of 12 donors also exhibited neutralization activity against at least one of H5N1 pseudotypes. Among them post-immune sera from 3 donors (Subject #1, #2 and #4) have low neutralization titers (IC50 ranging from 1∶10 to 1∶160) against all 6 H5N1 pseudotypes. Compared to the pre-immune sera, neutralization titers in post-immune sera increased at least 4 folds or higher (
For the comparison, neutralization titers were also measured by a standard MN assay against two representative H5N1 strains (A/Cambodia/P0322095/05 and A/Shenzhen/406H/06) and H1N1 strain (A/WSN/33). Before 2008–2009 seasonal flu vaccination, sera from all 12 donors exhibited no detectable levels of neutralization titers against all three strains; while after 2008–2009 seasonal flu vaccination, sera from 8 of 12 donors exhibited neutralization activity at the serum dilution from 1∶10 to 1∶160 against H1N1 strain; but still none of them have measurable neutralization activity against 2 H5N1 strains (
H1N1 | H5N1 | |||
Human subjects | A/WSN/33 | A/Cambodia/P0322095/05 | A/Shenzhen/406H/06 | |
Pre- | 1 | <1∶10 |
<1∶10 | <1∶10 |
immune | 2 | <1∶10 | <1∶10 | <1∶10 |
sera | 3 | <1∶10 | <1∶10 | <1∶10 |
4 | <1∶10 | <1∶10 | <1∶10 | |
5 | <1∶10 | <1∶10 | <1∶10 | |
6 | <1∶10 | <1∶10 | <1∶10 | |
7 | <1∶10 | <1∶10 | <1∶10 | |
8 | <1∶10 | <1∶10 | <1∶10 | |
9 | <1∶10 | <1∶10 | <1∶10 | |
10 | <1∶10 | <1∶10 | <1∶10 | |
11 | <1∶10 | <1∶10 | <1∶10 | |
12 | <1∶10 | <1∶10 | <1∶10 | |
Post- | 1 | 1∶40 | <1∶10 | <1∶10 |
immune | 2 | <1∶10 | <1∶10 | <1∶10 |
sera | 3 | <1∶10 | <1∶10 | <1∶10 |
4 | <1∶10 | <1∶10 | <1∶10 | |
5 | 1∶10 | <1∶10 | <1∶10 | |
6 | 1∶10 | <1∶10 | <1∶10 | |
7 | 1∶40 | <1∶10 | <1∶10 | |
8 | <1∶10 | <1∶10 | <1∶10 | |
9 | 1∶10 | <1∶10 | <1∶10 | |
10 | 1∶10 | <1∶10 | <1∶10 | |
11 | 1∶160 | <1∶10 | <1∶10 | |
12 | 1∶10 | <1∶10 | <1∶10 |
*<1∶10: not detected.
We next performed PN release/entry assay to determine the effect of pre- and post-human immune sera on pseudotype release. To accomplish this, 293T cells were first co-transfected with pHR'CMV-Luc, pCMVΔR8.2, one of two CMV/R-H5HA (A/Cambodia/P0322095/05 and A/Shenzhen/406H/06) and CMVR-N1NA or with pHR'CMV-Luc, pCMVΔR8.2, and CMVR-VSV-G control as described before
H5N1 | ||||
Human subjects | VSV | A/Cambodia/P0322095/05 | A/Shenzhen/406H/06 | |
Pre- | 1 | <1∶10 |
1∶40 | 1∶40 |
immune | 2 | <1∶10 | 1∶40–1∶160 | 1∶10 |
sera | 3 | <1∶10 | 1∶40–1∶160 | 1∶40–1∶160 |
4 | <1∶10 | 1∶40–1∶160 | 1∶10–1∶40 | |
5 | <1∶10 | <1∶10 | 1∶10–1∶40 | |
6 | <1∶10 | <1∶10 | 1∶40 | |
7 | <1∶10 | 1∶40 | <1∶10 | |
8 | <1∶10 | <1∶10 | <1∶10 | |
9 | <1∶10 | 1∶10 | 1∶10 | |
10 | <1∶10 | 1∶160–1∶640 | 1∶160 | |
11 | <1∶10 | <1∶10 | <1∶10 | |
12 | <1∶10 | <1∶10 | 1∶10–1∶40 | |
Post- | 1 | <1∶10 | >1∶640 |
1∶640 |
immune | 2 | <1∶10 | 1∶160–1∶640 | 1∶40 |
sera | 3 | <1∶10 | >1∶640 | 1∶160–1∶640 |
4 | <1∶10 | 1∶640 | 1∶160–1∶640 | |
5 | <1∶10 | 1∶160–1∶640 | 1∶40–1∶160 | |
6 | <1∶10 | 1∶640 | 1∶40–1∶160 | |
7 | <1∶10 | 1∶640 | 1∶160–1∶640 | |
8 | <1∶10 | 1∶10 | 1∶160 | |
9 | <1∶10 | 1∶40–1∶160 | 1∶40 | |
10 | <1∶10 | >1∶640 | 1∶160 | |
11 | <1∶10 | 1∶10–1∶40 | 1∶40–1∶160 | |
12 | <1∶10 | 1∶40–1∶160 | 1∶40–1∶160 |
*<1∶10: not detected.
**>1∶640: the Ab titers greater than the highest antibody dilutions.
Before 2008–2009 seasonal influenza vaccination, sera from 7 of 12 donors exhibited low, but measurable neutralization titers (IC50 ranging from 1∶40 to 1∶640 serum dilution) against H5N1 pseudotype A/Cambodia/P0322095/05 and sera from 9 of 12 donors exhibited low, but measurable neutralization titers (IC50 ranging from 1∶10 to 1∶160 serum dilution) against H5N1 pseudotype A/Shenzhen/406H/06, while none of them exhibited detectable levels of neutralization activity against VSV-G control. After 2008–2009 seasonal influenza vaccination, sera from 11 of 12 donors exhibited at least 4 fold increased neutralization titers against H5N1 pseudotypes A/Cambodia/P0322095/05 and A/Shenzhen/406H/06. But still none of them exhibited detectable levels of neutralization activity against VSV-G control. Sera from remaining 1 donor (Subject #10) that did not exhibit increased neutralization titers after 2008–2009 seasonal influenza vaccination actually had the highest neutralization titers in the pre-immune serum samples. Thus, these data indicate that low, but measurable levels of neutralization activity against H5N1 virus release exists even before seasonal influenza vaccination. However, after seasonal influenza vaccination, neutralization activity against H5N1 virus release significantly increases.
To better understand the contribution of anti-HA versus anti-NA antibodies in immune sera to virus release and to virus entry, we generated two immune sera against HA and NA, respectively, by immunizing female BALB/c mice with DNA plasmids expressing H5HA and N1NA, respectively. We then measured their neutralization activity by both PN entry and PN release/entry assays.
a) Titration of anti-HA immune sera by PN entry and PN release/entry assays; b) Titration of anti-NA immune sera by PN entry and PN release/entry assays. Black close square stands for anti-H5N1 neutralizing antibody response measured by PN release/entry assay; Red close square stands for anti-H5N1 neutralizing antibody response measured by PN entry assay; Black open circle stands for anti-VSV-G neutralizing antibody response measured by PN release/entry assay; Red open circle stands for anti-VSV-G neutralizing antibody response measured by PN entry assay.
Having demonstrated that low levels of heterosubtypic neutralizing antibody response against HPAI H5N1 are indeed elicited with seasonal influenza vaccine in humans, we next tested if such antibody response can also be elicited in mice and if so whether such antibody response offers any immune protection against lethal challenge of HPAI H5N1 viruses. To test this, we carried out two immunization and challenge experiments. In the first experiment female BALB/c mice randomly divided into three groups (5 mice per group) were injected i.m. twice with PBS, VLP expressing H5HA and N1NA or the seasonal influenza vaccine, respectively, followed by the challenge of 10 MLD50 HPAI H5N1 A/Shenzhen/406H/06 viruses. Dose 10 MLD50 was chosen to ensure 100% mortality rates in PBS control. In the second experiment 2 mice were injected i.m. twice with PBS, 2 mice with VLP and 22 mice with seasonal influenza vaccine followed by the same H5N1 challenge. Because both experiments showed similar trends of protection efficacy, we combined data of both experiments for analyses.
a) Mean and standard deviation of percentage of original body weight in three groups as well as two individual mice (#2 and #25) after challenged with HPAI H5N1 virus. Black line: PBS control group; red line: VLP group; blue line: seasonal influenza vaccine group; purple line: mouse #2 vaccinated with seasonal influenza vaccine; green line: mouse #25 vaccinated with seasonal influenza vaccine. b) Survival rates after challenged with HPAI H5N1 virus. Survival rate was calculated based on percent survival within each experimental group. Black line: PBS control group; red line: VLP group; blue line: seasonal influenza vaccine group. The data shown here were the combined results of two individual experiments. The survival curves between different groups were compared by the log-rank test.
To better understand the immune protection, we compared neutralizing antibody titers in post-boost immune sera in individual mice against H1N1 A/Brisbane/59/2007, H3N2 A/Brisbane/10/2007 and H5N1 A/Shenzhen/406H/06 pseudotypes using the PN entry assay. As expected, all mice received PBS did not have any neutralizing antibody activity against all three pseudotypes (
PBS: sera from individual PBS control mouse group; VLP: immune sera from individual mice immunized with H5N1 VLP; Vaxigrip: immune sera from individual mice immunized with seasonal influenza vaccine. The data shown here were the combined results of two individual experiments.
To further determine whether the levels of heterosubtypic neutralizing antibody responses elicited with seasonal influenza vaccine are statistically significant, we compared neutralization activity of immune sera among the three groups.
Horizontal bars show the mean values of percentage inhibition in each of three groups. The data shown here were the combined results of two individual experiments.
Heterosubtypic neutralizing antibody response between the annual seasonal influenza vaccine and other potential pandemic influenza viruses such as HPAI H5N1 viruses has important implications for public health pandemic influenza preparedness. In the present study, we demonstrated that in most human individuals 2008–2009 seasonal influenza vaccination elicited low, but measurable, heterosubtypic neutralization activity against various clades and subclades of H5HA and this heterosubtypic neutralization activity can be detected by a sensitive PN assay, but not by MN assay. Thus, on one hand our results (
The PN assay is not only more sensitive than conventional MN and HI assays, but also allows us to dissect whether the neutralizing antibody responses act on virus release or virus entry or both (see
A number of studies in animals have shown that cross-protective immunity against H5N1 can be elicited with seasonal influenza vaccines or infection
Although these aforementioned studies strongly suggested that antibody responses elicited with seasonal influenza vaccines confer certain degree of protection against heterosubtypic H5N1 challenge, what neutralizing antibody titers were required for such protection was not clear. In the present study, the sensitive PN assay enables us to detect low levels of heterosubtypic neutralizing antibody responses in mice elicited with seasonal influenza vaccine (
Finally, in the present study we also showed that in most human cases immune sera elicited with seasonal influenza vaccination had several fold higher heterosubtypic neutralizing antibody responses against H5N1 than those found in two survived mice mentioned above (see
We thank all human volunteers who participated in this study. We also thank Dr. L. Naldini at the University Torino Medical School, Torino, Italy for lentiviral vectors, Dr. T. Toyoda at the Pasteur Institute of Shanghai for the H1HA construct from the A/WSN/33 strain, Dr. Boping Zhou at the Donghu Infectious Hospital for the H5N1 strain (A/Shenzhen/406H/06).