Long-Term Immunogenicity of an Inactivated Split-Virion 2009 Pandemic Influenza A H1N1 Virus Vaccine with or without Aluminum Adjuvant in Mice

In 2009, a global epidemic of influenza A(H1N1) virus caused the death of tens of thousands of people. Vaccination is the most effective means of controlling an epidemic of influenza and reducing the mortality rate. In this study, the long-term immunogenicity of influenza A/California/7/2009 (H1N1) split vaccine was observed as long as 15 months (450 days) after immunization in a mouse model. Female BALB/c mice were immunized intraperitoneally with different doses of aluminum-adjuvanted vaccine. The mice were challenged with a lethal dose (10× 50% lethal dose [LD₅₀]) of homologous virus 450 days after immunization. The results showed that the supplemented aluminum adjuvant not only effectively enhanced the protective effect of the vaccine but also reduced the immunizing dose of the vaccine. In addition, the aluminum adjuvant enhanced the IgG antibody level of mice immunized with the H1N1 split vaccine. The IgG level was correlated to the survival rate of the mice. Aluminum-adjuvanted inactivated split-virion 2009 pandemic influenza A H1N1 vaccine has good immunogenicity and provided long-term protection against lethal influenza virus challenge in mice.     

Persistence of Immunogenicity of a Monovalent Influenza Virus A/H1N1 2009 Vaccine in Healthy Volunteers

After WHO declared H1N1 pandemic, global vaccination was carried out immediately after much research. However, the data on long-term immunogenicity were lacking. We aimed to investigate the long-term immunogenicity of different H1N1 vaccine dosage groups 24 weeks after vaccination by a randomized clinical trial. A total of 218 participants were stratified into adult (≤60 years old) and elderly (>60 years old) groups. The adults were randomized in a 1:1:1 ratio. The first group received a single dose of vaccine with 15 μg hemagglutination antigen (HA). The other two groups received two doses with 15 μg or 30 μg HA triweekly. The elderly were randomized 1:1 for two doses of 15 or 30 μg HA. We evaluated serologic responses at prevaccination and weeks 3, 6, and 24. We also examined possible associated factors of immunogenicity by multivariate logistic regression analyses. At week 24, seroprotection (anti-HA antibody level ≥ 1:40) remained at 76.8% and 46.2% in the adult and elderly groups, respectively. The adult group had a higher seroprotection rate (odds ratio of 2.98, 95% confidence interval [CI]: 1.21 to 7.36) than the elderly group. There was no statistical difference in seroprotection and seroconversion rates between different adult and elderly dosage groups. Lower immunogenicity in the elderly than in the adults 24 weeks after the vaccination was observed. However, there was no statistically significant difference among different dose groups. Therefore, we suggest only a single vaccination dose of 15 μg HA for adults and two doses of 15 μg HA for the elderly in the future.     

Humans and Ferrets with Prior H1N1 Influenza Virus Infections Do Not Exhibit Evidence of Original Antigenic Sin after Infection or Vaccination with the 2009 Pandemic H1N1 Influenza Virus

The hypothesis of original antigenic sin (OAS) states that the imprint established by an individual''s first influenza virus infection governs the antibody response thereafter. Subsequent influenza virus infection results in an antibody response against the original infecting virus and an impaired immune response against the newer influenza virus. The purpose of our study was to seek evidence of OAS after infection or vaccination with the 2009 pandemic H1N1 (2009 pH1N1) virus in ferrets and humans previously infected with H1N1 viruses with various antigenic distances from the 2009 pH1N1 virus, including viruses from 1935 through 1999. In ferrets, seasonal H1N1 priming did not diminish the antibody response to infection or vaccination with the 2009 pH1N1 virus, nor did it diminish the T-cell response, indicating the absence of OAS in seasonal H1N1 virus-primed ferrets. Analysis of paired samples of human serum taken before and after vaccination with a monovalent inactivated 2009 pH1N1 vaccine showed a significantly greater-fold rise in the titer of antibody against the 2009 pH1N1 virus than against H1N1 viruses that circulated during the childhood of each subject. Thus, prior experience with H1N1 viruses did not result in an impairment of the antibody response against the 2009 pH1N1 vaccine. Our data from ferrets and humans suggest that prior exposure to H1N1 viruses did not impair the immune response against the 2009 pH1N1 virus.     

Serologic Evidence of Pandemic Influenza Virus H1N1 2009 Infection in Cats in China

Infection of domestic cats with (H1N1) pandemic 2009 (pdm09) influenza A virus has recently been documented. In this paper, we report for the first time the sporadically current seroprevalence of (H1N1) pdm09 influenza A virus infection in cats in China. Thirteen of 1,080 sera were found positive by nucleoprotein (NP)-specific enzyme-linked immunosorbent assays (ELISAs) in different cat populations in southern China. It is very important to stress further surveillance of pandemic (H1N1) 2009 influenza A virus in cats in southern China.     

Assessment of Route of Administration and Dose Escalation for an Adenovirus-Based Influenza A Virus (H5N1) Vaccine in Chickens

Highly pathogenic avian influenza (HPAI) virus causes one of the most economically devastating poultry diseases. An HPAI vaccine to prevent the disease in commercial and backyard birds must be effective, safe, and inexpensive. Recently, we demonstrated the efficacy of an adenovirus-based H5N1 HPAI vaccine (Ad5.HA) in chickens. To further evaluate the potential of the Ad5.HA vaccine and its cost-effectiveness, studies to determine the minimal effective dose and optimal route of administration in chickens were performed. A dose as low as 10⁷ viral particles (vp) of adenovirus-based H5N1 vaccine per chicken was sufficient to generate a robust humoral immune response, which correlated with the previously reported level of protection. Several routes of administration, including intratracheal, conjunctival, subcutaneous, and in ovo routes, were evaluated for optimal vaccine administration. However, only the subcutaneous route of immunization induced a satisfactory level of influenza virus-specific antibodies. Importantly, these studies established that the vaccine-induced immunity was cross-reactive against an H5N1 strain from a different clade, emphasizing the potential of cross-protection. Our results suggest that the Ad5.HA HPAI vaccine is safe and effective, with the potential of cross-clade protection. The ease of manufacturing and cost-effectiveness make Ad5.HA an excellent avian influenza vaccine candidate with the ability to protect poultry from HPAI virus infection. Considering the limitations of the influenza vaccine technology currently used for poultry applications, any effort aimed at overcoming those limitations is highly significant.Influenza A virus is a segmented, negative-strand RNA virus that belongs to the family Orthomyxoviridae, which is divided into subtypes based on serological reactions of the two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Thus far, 16 different HA subtypes (H1 to H16) and 9 different NA subtypes (N1 to N9) (11, 34) have been identified. Each of the subtypes has been isolated from waterfowl species, the natural hosts of all known influenza A viruses. These birds are the reservoir for the spread of influenza virus worldwide in wild birds and poultry (19, 34). Avian influenza (AI) virus strains are further classified into low and highly pathogenic avian influenza (LPAI and HPAI, respectively) viruses based on their pathogenicity.Continued outbreaks of HPAI viruses of the H5 and H7 subtypes in poultry in Asia, Europe, Africa, and Canada represent a serious risk for animal and public health worldwide. Avian influenza is one of the greatest concerns for public health that has emerged from an animal reservoir in recent times. Since the late 1990s, the number of outbreaks of avian influenza in poultry has dramatically increased. For example, in 2008-2009, 2,770 outbreaks occurred in Vietnam, 1,143 in Thailand, 1,084 in Egypt, and 219 in Turkey; outbreaks have also occurred in many other countries worldwide (http://www.oie.int/eng/en_index.htm).In 2008-2009, 20 million poultry died or were depopulated because of HPAI outbreaks. This had devastating consequences for the international poultry industry and raised concerns about the potential for transmission to humans and the possible pandemic spread of lethal disease. Culling represents the first line of defense against avian influenza virus; however, continuing outbreaks over the last 6 years revealed that implementation of culling at the farm level was insufficient to halt the spread of disease.Vaccines, in conjunction with other measures of prevention and management, may represent an alternative to preemptive culling to achieve a reduction in the rate of transmission by reducing the susceptibility of healthy flocks at risk. Although vaccination has been recommended by the World Organization for Animal Health (OIE) and the Food and Agriculture Organization (FAO) to control AI, few effective AI vaccines are available (http://www.oie.int/eng/avian_influenza/OIE_FAO_Recom_05.pdf). Conventional inactivated vaccines containing the same viral subtype as field virus (with differing degrees of antigenic similarity) (4, 5, 22), inactivated vaccines generated through reverse genetic techniques (18, 33), and recombinant vaccines (3, 15, 21, 23) have been tested. However, production constraints associated with conventional inactivated influenza virus vaccines that are manufactured in eggs could severely hinder control of an emerging AI virus with pandemic potential (7).We investigated recombinant replication-defective adenoviruses as possible influenza vaccine vehicles for poultry. Recombinant adenovirus-based vaccines are highly effective inducers of both humoral immunity and cellular immunity in mammals and have shown promise as vaccine vehicle candidates against numerous infectious pathogens (2, 12, 14, 24, 32). Previously, we generated Ad5.HA, an E1/E3-deleted human adenovirus serotype 5 (Ad5)-based vector that expresses the codon-optimized hemagglutinin (HA) gene from the influenza A/Vietnam/1203/04 (H5N1) virus (13). The Ad5.HA vaccine induced humoral and cellular immune responses against HA and protected against influenza virus challenges in both mice and chickens. We have now extended our studies to determine the efficacy of Ad5.HA immunization in chickens when administered at different dosages via different routes.     

Immunologic Characterization of a Rhesus Macaque H1N1 Challenge Model for Candidate Influenza Virus Vaccine Assessment

Despite the availability of annually formulated vaccines, influenza virus infection remains a worldwide public health burden. Therefore, it is important to develop preclinical challenge models that enable the evaluation of vaccine candidates while elucidating mechanisms of protection. Here, we report that naive rhesus macaques challenged with 2009 pandemic H1N1 (pH1N1) influenza virus do not develop observable clinical symptoms of disease but develop a subclinical biphasic fever on days 1 and 5 to 6 postchallenge. Whole blood microarray analysis further revealed that interferon activity was associated with fever. We then tested whether type I interferon activity in the blood is a correlate of vaccine efficacy. The animals immunized with candidate vaccines carrying hemagglutinin (HA) or nucleoprotein (NP) exhibited significantly reduced interferon activity on days 5 to 6 postchallenge. Supported by cellular and serological data, we conclude that blood interferon activity is a prominent marker that provides a convenient metric of influenza virus vaccine efficacy in the subclinical rhesus macaque model.     

Development of an Immunochromatographic Assay Specifically Detecting Pandemic H1N1 (2009) Influenza Virus

The pandemic caused by a new type of influenza virus, pandemic H1N1 (2009) influenza virus A (AH1pdm), has had a major worldwide impact. Since hemagglutinin (HA) genes are among the most specific genes in the influenza virus genome, AH1pdm can be definitively diagnosed by viral gene analysis targeting the HA genes. This type of analysis, however, cannot be easily performed in clinical settings. While commercially available rapid diagnosis kits (RDKs) based on immunochromatography can be used to detect nucleoproteins (NPs) of influenza A and B viruses in clinical samples, there are no such kits that are specific for AH1pdm. We show here that an RDK using a combination of monoclonal antibodies against NP can be used to specifically detect AH1pdm. The RDK recognized AH1pdm virus isolates but did not recognize seasonal H1N1 and H3N2 and influenza B viruses, indicating that the specificity of the RDK is 100%. A parallel comparison of RDK with a commercial influenza A/B virus kit revealed that both types of kits had equal sensitivities in detecting their respective viruses. Preliminary evaluation of clinical samples from 5 individuals with PCR-confirmed human AH1pdm infection showed that the RDK was positive for all samples, with the same detection intensity as that of a commercial influenza A/B virus kit. This RDK, together with a new vaccine and the stockpiling of anti-influenza drugs, will make aggressive measures to contain AH1pdm infections possible.The pandemic caused by a new type of influenza virus, pandemic H1N1 (2009) influenza virus A (AH1pdm), has had a major worldwide impact. As of 27 September 2009, more than 4,100 deaths from AH1pdm infection have been reported to the World Health Organization (WHO) (http://www.who.int/csr/don/2009_10_02/en/index.html). Current methods used to diagnose AH1pdm virus in clinical specimens are based on viral RNA analysis targeting hemagglutinin (HA) genes, because the HA genes are among the most specific genes in the influenza virus genome. Although these methods are highly sensitive, they usually take more than 2 to 6 h to complete and require well-equipped laboratories with virologists or well-trained medical technicians and specialized tools for virus genome isolation and amplification (6, 8) (http://www.who.int/entity/csr/resources/publications/swineflu/CDCRealtimeRTPCR_SwineH1Assay-2009_20090430.pdf). Rapid diagnostic kits (RDKs) based on immunochromatography utilize antibodies (Abs) against antigens of interest. Although RDKs are usually less sensitive than genetic assays, they do not require the isolation of a viral genome, thus overcoming the intrinsic difficulties of viral gene analyses. RDKs for many infectious diseases (2, 4, 9, 11-14), including influenza viruses A and B (1), are commercially available. However, RDKs capable of distinguishing AH1pdm viruses from seasonal influenza viruses have yet to be implemented in a clinical setting.Nucleoproteins (NPs) of influenza A, B, and C viruses have important differences in their antigenicities that enable them to be distinguished from one another but are highly conserved within each major serotype. Thus, antibodies to NPs have been utilized in commercially available RDKs to distinguish between influenza A and B viruses (15). In a monoclonal antibody (MAb) preparation procedure targeting NPs derived from highly pathogenic H5N1 avian influenza (HPAI), we obtained 2 MAbs that reacted with NPs of AH1pdm as well as that of HPAI but not those of seasonal influenza A virus. We have therefore utilized these MAbs in the development of novel RDKs for AH1pdm, and we have validated these RDKs in laboratory environments.     

Immunogenicity Associated with the Routine Use of an Influenza A H1N1 Vaccine in Health Care Personnel in Guangzhou,China

We present immunogenicity data on the routine vaccination of 103 health care personnel during the 2009 H1N1 national vaccination campaign. The seroprotection rate (percentage of samples with hemagglutination inhibition titers of ≥1:40) was 83.2% at 30 days postvaccination, lower than those obtained in previously published controlled trials. Low baseline antibody levels and an increase in seroprotection in a negative-control cohort suggest that the virus remains prevalent.The 2009 swine-origin influenza A H1N1 virus pandemic has caused over 15,000 laboratory-confirmed deaths (9). While this is lower than original estimates, transmission continues and the threat of a second wave of infections by a more virulent strain remains. Many countries have already identified severe cases of influenza related to a mutated H1N1 strain (8). Measures for prevention of any further spread of pandemic influenza must be taken.The most effective measure for prevention of the spread of influenza is mass vaccination. This not only confers primary immunity but also greatly reduces the replication capacity of the virus in the host, thereby decreasing the opportunity for genetic mutation and antigen drift. Health care personnel (HCP) are a high-priority group for vaccination campaigns because of their interaction with patients, who may be sick with the disease or may be particularly susceptible to infection (2). While previous double-blind controlled trials have shown the potential effectiveness of the 2009 H1N1 vaccine, there have been no studies on its routine use and effectiveness (6). Furthermore, there have been no studies on the baseline levels of H1N1 immunity or the immunogenicity of the vaccine in Guangzhou, where the first cases of H1N1 were identified in the People''s Republic of China (PRC). We present immunogenicity data on the routine use of the vaccine in a population of HCP at the Guangzhou Center for Disease Control (CDC) in China.One hundred three HCP presenting for vaccination were enrolled on a rolling and volunteer basis and were administered the vaccine by use of standard procedures (4). Information about previous vaccination with the seasonal vaccine and known influenza-like illness within the last 6 months was recorded. Patients were excluded if they had already received the H1N1 vaccine or if they had received any vaccination in the last 6 weeks. All patients provided written informed consent. The resulting group of participants was composed of 56 males and 47 females, aged 19 to 55 years, all from the Yuexiu district, Guangzhou City, China. Blood samples were collected prior to the vaccination (T₀) and at 15 (T₁₅) and 30 (T₃₀) days after the vaccination. A cohort of 145 HCP was enrolled as a negative control, and blood samples were collected before and after the study period. The hemagglutinin inhibition (HI) test was performed on serial 2-fold dilutions of each blood sample (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Reverse cumulative distribution curves of hemagglutination inhibition titers in both a vaccinated cohort (T₀, T₁₅, and T₃₀) and a negative-control cohort (Control 1 and Control 2). Values are expressed as a reciprocal of the dilution. T₀, day 0; T₁₅, day 15 after vaccination; T₃₀, day 30 after vaccination; Control 1, negative control before study period; Control 2, negative control after study period.The influenza A H1N1 monovalent, split-virus, nonadjuvant vaccines were supplied by the Ministry of Health, People''s Republic of China, and manufactured by Tianyuan Bio-Pharmaceutical Co., Ltd. (batch number 20090902), through the nationwide vaccination program. Each 0.5-ml dose contained 15 μg hemagglutinin, as prescribed by national guidelines.Serum HI antibody titers were evaluated by observing the detectable HI titer (≥1:10), seroprotection rate (percentage of samples with HI titers of ≥1:40), and seroconversion rate (percentage of samples with 4-fold increases in HI titer and HI titers of ≥1:40) and the geometric mean titer (GMT) (1). For GMT calculations, antibody levels below the detection limit (<1:10) were assigned the value of 1:5. All values are reported with 95% confidence intervals (CIs). GMT calculations were log transformed prior to statistical tests to account for skewed distribution. All P values are two tailed.Of the 103 participants, 7 failed to return on day 15 and 8 failed to return on day 30. Therefore, 103, 96, and 95 patient samples were received for each time point (T₀, T₁₅, and T₃₀). No adverse side effects or flu-like symptoms were reported for these eight patients.Pre- and postimmunization HI titers are shown in Table ​Table1.1. At the baseline (T₀), 10 patients (9.7%) had detectable titers of antibody against the pandemic H1N1 vaccine strain. Five patients (4.9%) had antibody titers that were greater than 1:40.

TABLE 1.

Reciprocal HI titers, seropositive rates, seroprotection rates, and seroconversion rates at the baseline and at days 15 and 30 in healthy HCP immunized with the 2009 pandemic influenza A H1N1 vaccine^a

Cardiac Tamponade and Heart Failure Due to Myopericarditis as a Presentation of Infection with the Pandemic H1N1 2009 Influenza A Virus

We describe a fatal case of myopericarditis presenting with cardiac tamponade in a previously healthy 11-year-old child. Pandemic H1N1 2009 influenza A virus sequences were identified in throat and myocardial tissues and pericardial fluid, suggesting damage of myocardial cells directly caused by the virus.     

Influenza and HIV: Lessons from the 2009 H1N1 Influenza Pandemic

Influenza is a common respiratory disease in adults, including those infected with HIV. In the spring of 2009, a pandemic influenza A (H1N1) virus (pH1N1) emerged. In this article, we review the existing literature regarding pH1N1 virus infection in HIV-infected adults, which suggests that susceptibility to pH1N1 virus infection and severity of influenza illness are likely not increased in HIV-infected adults without advanced immunosuppression or comorbid conditions. The risk of influenza-related complications, however, may be increased in those with advanced immunosuppression or high-risk comorbid conditions. Prevention and treatment of high-risk comorbid conditions and annual influenza vaccination should continue to be part of HIV clinical care to help prevent influenza illness and complications. Additional information about pH1N1 vaccine immunogenicity and efficacy in HIV-infected patients would be useful to guide strategies to prevent influenza virus infection in this population.     

Utility of Pandemic H1N1 2009 Influenza Virus Recombinant Hemagglutinin Protein-Based Enzyme-Linked Immunosorbent Assay for Serosurveillance

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies against the pandemic H1N1 2009 influenza A virus, employing a recombinant hemagglutinin protein of the virus, was compared to the hemagglutination inhibition (HI) test using 783 serum samples. The results showed a concordance of 98.4%, suggesting the utility of the ELISA in serosurveillance. Two hundred sixty-nine (100%) serum samples with an HI titer of ≥20 were ELISA reactive.Influenza viruses are negative-strand RNA viruses that belong to the family Orthomyxoviridae, which includes 4 genera, Influenzavirus A, B, and C and Thogotovirus. Influenza A viruses are widely distributed in nature and can infect a wide variety of mammals and birds. Based on the antigenicity of the two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), influenza A viruses have been classified into 16 HA and 9 NA subtypes. Of these, H1, H2, and H3 HA subtypes and N1 and N2 NA subtypes have circulated in human populations. In recent years, H5N1 virus of avian origin was expected to be a pandemic-causing pathogen (4). However, the first pandemic of this century was caused by a novel H1N1 influenza A virus of swine origin that emerged in 2009 (hereinafter called p-H1N1-09) (3, 5).Due to the circulation of several influenza A virus subtypes, cross-reactivity is a major problem in influenza virus serology. The inhibition of hemagglutination (HI) caused by antibodies to the HA of the virus is routinely used for assessing the prevalence of a specific virus in a community or an animal population.India was affected by the pandemic H1N1 influenza during the latter half of 2009 (2). In order to understand the degree of exposure of different populations to the virus, an extensive serosurvey was undertaken by the National Institute of Virology, Pune, India. The test of choice, HI, was performed as described earlier (1). Considering the requirement of fresh red blood cells and time and the cumbersome protocol, it was thought important to evaluate the utility of a recombinant HA protein enzyme-linked immunosorbent assay (ELISA) for the detection of p-H1N1-09 IgG antibodies as evidence of exposure to this novel virus.The HA gene of the p-H1N1-09 influenza virus isolated at the National Institute of Virology (A/India-Blore/NIV310/2009, GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GU292347","term_id":"273039286","term_text":"GU292347"}}GU292347) was PCR amplified, cloned into the pFastBac1 vector (Invitrogen) within the EcoRI and XhoI restriction sites, and expressed with a baculovirus expression system (Invitrogen) in an insect cell line. The sequence of the cloned HA was identical to that of the original isolate. The HA protein was purified by lentil lectin affinity chromatography (GE Healthcare) and used for ELISA.An indirect sandwich ELISA was performed. Briefly, a Maxisorb microtiter plate (Nunc) was coated with p-H1N1-09 HA protein, 2 μg/well, and incubated at 37°C for 2 h. The plate was blocked with phosphate-buffered saline (PBS) containing 10% donor calf serum, 0.5% Tween 20, 0.5% gelatin (blocking solution) at 37°C for 30 min. After washing 3 times with the wash solution (PBS containing 0.5% Tween 20), test serum samples and positive and negative controls diluted 1:100 in blocking solution were added to the previously designated wells of the coated assay plates and incubated at 37°C for 30 min. Following 4 washes with the wash solution, horseradish peroxidase-conjugated anti-human IgG (Sigma Chemicals, St. Louis, MO) was added to each well as the detector antibody and allowed to incubate for 30 min. The enzymatic reaction with the substrate (O-phenylenediamine and urea peroxide, 10 min) was stopped by the addition of 4 M H₂SO₄, and optical density (OD) values were determined at 492 nm. Human serum samples known to be positive and negative for HI antibodies against the pandemic influenza virus were included in every assay plate as controls. The cutoff values for IgG anti-p-H1N1-09 antibodies were calculated as the mean OD values for the results of 3 negative controls in triplicate. Samples with values greater than or equal to the cutoff values were considered antibody positive. Samples showing OD values within 10% of the cutoff value were considered borderline reactive and repeated.A total of 783 serum samples previously screened by the HI test for the presence of p-H1N1-09 antibodies were retested in the ELISA. As evident from the results (Table ​(Table1),1), the ELISA emerged as an excellent assay for the detection of virus-specific antibodies. Of the 397 HI-negative samples, 389 were scored negative in the ELISA, giving 98% specificity in the comparison to the gold-standard HI test. Importantly, all samples with HI titers of >20 (n = 269) were positive in the ELISA, documenting 100% sensitivity of the ELISA. A large number of samples (n = 117) exhibited low HI titers (1:10). Usually, reactivity at this dilution is not considered specific. Of these, only 4 were recorded as reactive in the ELISA. When HI-negative samples with titers of 10 were considered antibody negative, the ELISA specificity was 97.7% and the concordance between the HI and ELISA results was 98.5%. To examine the relationship of the HI and ELISA titers, 2-fold dilutions of the test samples were tested in the ELISA. The reciprocal of the highest dilution at which the OD value was greater than or equal to the cutoff value was considered the IgG anti-p-H1N1-09 HA titer of the serum. All HI-positive sera with titers ≥20 were titrated in the ELISA. When the HI and ELISA titers were compared, the Spearman''s rank correlation coefficient was estimated to be 0.864. Thus, a good correlation between the HI and ELISA titers was apparent. In addition, a linear relationship was noted when the log HI and ELISA titers were compared (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Relationship of log HI and ELISA titers. Each point represents the mean positive ELISA titer for a given HI titer value. Error bars represent standard errors of the means.

TABLE 1.

Relationship of HI titers and IgG positivity in ELISA against p-H1N1-09 virus