Immunogenicity of influenza and HSV-1 mixed antigen ISCOMs in mice

Summary Immunostimulating complexes (ISCOMs) were prepared with mixtures of antigens from influenza A virus (A/PR/8/34 or A/Sichuan/2/87) and herpes simplex virus type 1 (HSV-1), and were characterised by enzyme linked immunosorbent assay (ELISA) and electron microscopy using double-labelling immunogold techniques employing monoclonal antibodies to influenza or HSV-1 glycoproteins. The immunogenicity of the mixed antigen ISCOMs was evaluated in mice, following administration by the subcutaneous route, by measuring the total and subclass IgG antibody responses. Protection of these animals against challenge with live influenza A/Sichuan virus or live HSV-1, was compared with that induced by immunization with aqueous mixed antigen preparations. It was found that relatively high humoral responses to both influenza and HSV antigens, and increased levels of protection to both influenza and HSV viruses were elicited in mice receiving the mixed antigen ISCOM preparation compared to those observed in animals receiving the mixed aqueous subunit preparation. The findings also indicate that antigens from more than one virus can be used in an ISCOM formulation to produce immunity and protection.     

Secretory immunological response after intranasal inactivated influenza A virus vaccinations: evidence for immunoglobulin A memory.

An intranasal, inactivated trivalent influenza A vaccine containing 7 micrograms of A/Bangkok/1/79 (H3N2) hemagglutinin was administered to 20 children aged 1 to 6 years to assess the local and systemic immune responses to antigen delivered to the respiratory tract. Six children without prior influenza virus infection exhibited no local immune response and manifested only a minimal systemic response to the intranasal vaccine. In contrast, five individuals who were previously infected with a live attenuated influenza A H3N2 virus vaccine, although having no residual secretory antibody at the time of challenge, promptly developed a local antibody response to intranasal, inactivated antigen. Therefore, the live influenza A virus vaccine had induced memory in the local immunoglobulin A (IgA) immune system. The third group of nine children had previously been infected with wild-type H3N2 influenza virus. A majority of these children had residual local and systemic antibody at the time of challenge but they demonstrated some boosting of local IgA antibody with administration of intranasal inactivated vaccine. The competence of the secretory IgA immune system in young children in mounting primary and secondary responses to influenza antigens has important implications for approaches to prevention of influenzal illness.     

Development and persistence of local and systemic antibody responses in adults given live attenuated or inactivated influenza A virus vaccine.

An enzyme-linked immunosorbent assay was used to measure nasal-wash and serum isotype-specific hemagglutinin antibody responses in 109 seronegative (hemagglutination-inhibiting titer less than or equal to 1:8) adults vaccinated intranasally with live attenuated A/Washington/897/80 (H3N2) or A/California/10/78 (H1N1) cold-adapted (ca) virus or with licensed subvirion vaccine subcutaneously. Live and inactivated virus elicited serum immunoglobulin A (IgA) responses in 83 and 96% of vaccinees, respectively, and elicited serum IgG responses in 72 and 100% of vaccinees. Inactivated virus induced higher titers of serum antibodies than did live virus and stimulated a nasal-wash IgG response more often than did live virus (94 versus 59%, P less than 0.01). In contrast, only 38% of inactivated virus vaccinees had local IgA responses compared with 83% of live virus vaccinees. Serum IgA and IgG and nasal IgG antibody titers remained elevated above prevaccination levels for at least 6 months in most of the live and inactivated vaccine responders, but the mean level of local IgA antibody induced by infection with live virus vaccine, in particular, decreased substantially. Considered in the context of previous work, the finding that live virus vaccine induced relatively long-lasting antibody in both local and serum compartments suggested that this vaccine may be a suitable alternative to inactivated vaccine for use in healthy persons.     

In elderly persons live attenuated influenza A virus vaccines do not offer an advantage over inactivated virus vaccine in inducing serum or secretory antibodies or local immunologic memory.

In a double-blind, randomized trial, 102 healthy elderly subjects were inoculated with one of four preparations: (i) intranasal bivalent live attenuated influenza vaccine containing cold-adapted A/Kawasaki/86 (H1N1) and cold-adapted A/Bethesda/85 (H3N2) viruses; (ii) parenteral trivalent inactivated subvirion vaccine containing A/Taiwan/86 (H1N1), A/Leningrad/86 (H3N2), and B/Ann Arbor/86 antigens; (iii) both vaccines; or (iv) placebo. To determine whether local or systemic immunization augmented mucosal immunologic memory, all volunteers were challenged intranasally 12 weeks later with the inactivated virus vaccine. We used a hemagglutination inhibition assay to measure antibodies in sera and a kinetic enzyme-linked immunosorbent assay to measure immunoglobulin G (IgG) and IgA antibodies in sera and nasal washes, respectively. In comparison with the live virus vaccine, the inactivated virus vaccine elicited higher and more frequent rises of serum antibodies, while nasal wash antibody responses were similar. The vaccine combination induced serum and local antibodies slightly more often than the inactivated vaccine alone did. Coadministration of live influenza A virus vaccine did not alter the serum antibody response to the influenza B virus component of the inactivated vaccine. The anamnestic nasal antibody response elicited by intranasal inactivated virus challenge did not differ in the live, inactivated, or combined vaccine groups from that observed in the placebo group not previously immunized. These results suggest that in elderly persons cold-adapted influenza A virus vaccines offer little advantage over inactivated virus vaccines in terms of inducing serum or secretory antibody or local immunological memory. Studies are needed to determine whether both vaccines in combination are more efficacious than inactivated vaccine alone in people in this age group.     

Mucosal immunization of CD4+ T cell-deficient mice with an inactivated virus induces IgG and IgA responses in serum and mucosal secretions

Immunoglobulin (Ig) class switching can occur in the absence of alphabeta+ or gammadelta+ T cells when mice are infected with certain live viruses, although CD4 T helper cells are believed to be essential for induction of a high-affinity antibody response and for efficient isotype switching from IgM to IgG and IgA production. However, little information is available about the immune responses after mucosal immunization of CD4+ T cell-deficient mice with inactivated virus. In this study, we show that intranasal immunization with formalin-inactivated influenza A/PR8/34 virus induces IgG and IgA responses in serum and IgA responses in mucosal secretions in CD4+ T cell-deficient mice. All four subclasses of IgG were produced. IgG1/IgG2a ratios were found to be from 1 to 1.75, indicating that both Th1 and Th2 immune responses are induced by the inactivated influenza virus. The sera and mucosal secretions were found to have neutralizing activity against influenza virus in vitro. In addition, the mucosally immunized CD4+ T cell-deficient mice were protected completely from challenge with a lethal dose of live, pathogenic influenza virus. To our knowledge, this is the first demonstration that mucosal immunization with an inactivated virus induces immune responses in serum and mucosal secretions in CD4+ T cell-deficient mice.     

Serum and nasal wash antibodies associated with resistance to experimental challenge with influenza A wild-type virus.

To identify immunological predictors of resistance to influenza A infection and illness, the immunological status of live and inactivated virus vaccines subsequently challenged with H1N1 or H3N2 wild-type virus was examined. We refer to prechallenge antibodies of vaccinees receiving live attenuated virus as infection induced and those receiving inactivated virus as inactivated vaccine induced. Inactivated vaccine-induced protection against wild-type virus infection or illness correlated with the level of neuraminidase-inhibiting antibody in serum, local hemagglutinin immunoglobulin G (IgG) (but not IgA) enzyme-linked immunosorbent assay antibody, and hemagglutination-inhibiting antibody in serum. In contrast, infection-induced resistance to wild-type virus infection correlated with local hemagglutinin IgA antibody and neuraminidase-inhibiting antibody in serum, but not with hemagglutination-inhibiting antibody in serum. These observations suggest that live vaccine virus infection-induced and inactivated vaccine-induced immunity may involve different compartments of the immune system; sufficient antibody in either serum or nasal secretions is capable of conferring resistance.     

Cross-protection in mice infected with influenza A virus by the respiratory route is correlated with local IgA antibody rather than serum antibody or cytotoxic T cell reactivity

Mice previously infected with an aerosol of A/Rec 31 influenza virus were strongly protected against an aerosol challenge with A/Vic influenza as judged by lung virus titers recovered 2 days after the challenge infection. Such complete homotypic immunity was not achieved by priming with live Rec 31 virus injected i.v. or UV-inactivated Rec 31 virus administered s.c. together with Al(OH)₃ and saponin. The reason for the superior protective effect of the natural infection was investigated. The protection induced by respiratory infection with Rec 31 virus was specific for influenza A viruses. It was not correlated with specific serum hemagglutination inhibition antibody titer or cross-reactive cytotoxic T (T_c) cell reactivity. Moreover, the transfer of splenic and lymphoid T cell populations with strong secondary T_c activity did not significantly reduce lung virus titers in recipient mice 3 days after infection. The protection however occurred in parallel with the presence of cross-reactive IgA antibody in the lung washings. It thus appears that local secretory IgA plays a causal role in the prevention of cross-infection by influenza A virus. Serum antibody and T_c cells, on the other hand, may be crucial for recovery from such infection. All mice primed with live Rec 31 virus, administered i.v. or by aerosol and expressing equally high levels of T_c reactivity, survived a lethal challenge with A/PR8 virus. The same challenge, however, killed half of the mice immunized s.c. with inactivated Rec 31 virus which induced only a low level of T_c reactivity.     

Systemic and local antibody responses in elderly subjects given live or inactivated influenza A virus vaccines.

Intranasal live attenuated cold-adapted (ca) influenza A/Kawasaki/9/86 (H1N1) reassortant virus and parenteral inactivated influenza A/Taiwan/1/86 (H1N1) virus were given alone or in combination to 80 ambulatory elderly subjects. An enzyme-linked immunosorbent assay was used to measure hemagglutinin-specific (HA) antibodies in serum and nasal wash specimens collected before vaccination and 1 and 3 months later. Serum immunoglobulin G (IgG) and nasal wash IgA HA responses were elicited in 56 and 20%, respectively, of 25 inactivated-virus vaccinees and in 67 and 48%, respectively, of 27 recipients of both vaccines but in only 36 and 25%, respectively, of 28 vaccinees given live virus alone. Inactivated virus, administered alone or with live virus vaccine, induced higher titers of serum antibody than did the live virus alone. In contrast, nasal IgA HA antibody was elicited more often and in greater quantity by the vaccine combination than by either vaccine alone. Despite these differences, the peak titers of local antibody mounted by each group of vaccinees were similar. By 3 months postvaccination, serum IgG and nasal IgA HA antibody titers remained elevated above prevaccination levels in 50 and 17%, respectively, of the inactivated-virus vaccinees and in 46 and 23%, respectively, of recipients of both vaccines but in only 19 and 7%, respectively, of the live-virus and systemic antibodies, if vaccinees. The finding that live ca influenza A virus induced short-lived local and systemic antibodies, if confirmed, suggests that live virus vaccination may not be a suitable alternative or adjunct to inactivated virus vaccination for the elderly.     

Carbohydrate biopolymers enhance antibody responses to mucosally delivered vaccine antigens

We have evaluated the ability of two carbohydrate biopolymers, chitosan and gellan, to enhance antibody responses to subunit influenza virus vaccines delivered to the respiratory tracts of mice. Groups of mice were vaccinated three times intranasally (i.n.) with 10 microg of purified influenza B/Panama virus surface antigens (PSAs), which consist of hemagglutinin (HA) and neuraminidase (NA), either alone or admixed with chitosan or gellan solutions. Separate groups were vaccinated subcutaneously (s.c.) with PSAs adsorbed to Alhydrogel or chitosan or gellan alone i.n. Serum antibody responses were determined by enzyme-linked immunosorbent assay (ELISA) for influenza virus-specific immunoglobulin G (IgG) and by HA inhibition (HAI) and NA inhibition (NAI) assays. The local respiratory immune response was measured by assaying for influenza virus-specific IgA antibody in nasal secretions and by enumerating nasal and pulmonary lymphocytes secreting IgA, IgG, and IgM anti-influenza virus-specific antibodies by enzyme-linked immunospotting (ELISPOT). When administered alone i.n., B/Panama PSA was poorly immunogenic. Parenteral immunization with B/Panama PSA with Alhydrogel elicited high titers of anti-B/Panama antibodies in serum but a very poor respiratory anti-B/Panama IgA response. In contrast, i.n. immunization with PSA plus chitosan stimulated very strong local and systemic anti-B/Panama responses. Gellan also enhanced the local and serum antibody responses to i.n. PSA but not to the same extent as chitosan. The ability of chitosan to augment the immunogenicity of influenza vaccines given i.n. was confirmed using PSA prepared from an influenza A virus (A/Texas H1N1).     

The immune response to influenza vaccines

Specific immunity to influenza is associated with a systemic immune response (serum haemagglutination inhibition antibody), local respiratory immune response (virus-specific local IgA and IgG antibodies in nasal wash), and with the cell-mediated immune response. Both inactivated and live influenza vaccines induce virus-specific serum antibody which can protect against infection with influenza virus possessing the same antigenic specificity. In the absence of serum antibodies, local antibodies in nasal wash are a major determinant of resistance to infection with influenza virus. In comparative studies in humans it was shown that nasal secretory IgA develops chiefly after immunization with live cold-adapted (CA) vaccine, but persistent nasal secretory IgG was detected in both CA live and inactivated vaccines. The origin of nasal wash haemagglutination inhibition (HI) antibodies is not completely known. Recently it was found that cytotoxic T-cells (CTL) play an important role in immunity against influenza and in clearance of influenza virus from the body. In primed humans, inactivated influenza vaccine stimulates a cross-reactive T-cell response, whereas the ability of inactivated vaccine to stimulate such immunity in unprimed humans has not been determined. Data on the T-cell response to live vaccine in humans are limited to the development of secondary T-cell responses in primed individuals vaccinated with a host-range (HR) attenuated vaccine. The data obtained have shown that immunity induced by inactivated influenza vaccines is presumably dependent on the stimulation of serum antibody. Live CA vaccines not only stimulate a durable serum antibody response, but also induce long-lasting local respiratory tract IgA antibody that plays an important role in host protection.     

Protection against influenza virus infection by intranasal administration of hemagglutinin vaccine with chitin microparticles as an adjuvant

Chitin in the form of microparticles (chitin microparticles, CMP) has been demonstrated to be a potent stimulator of macrophages, promoting T-helper-1 (Th1) activation and cytokine response. In order to examine the mucosal adjuvant effect of CMP co-administered with influenza hemagglutinin (HA) vaccine against influenza infection, CMP were intranasally co-administered with influenza HA vaccine prepared from PR8 (H1N1) virus. Inoculation of the vaccine with CMP induced primary and secondary anti-HA IgA responses in the nasal wash and anti-HA IgG responses in the serum, which were significantly higher than those of nasal vaccination without CMP, and provided a complete protection against a homologous influenza virus challenge in the nasal infection influenza model. In addition, CMP-based immunization using A/Yamagata (H1N1) and A/Guizhou (H3N2) induced PR8 HA-reactive IgA in the nasal washes and specific-IgG in the serum. The immunization with A/Yamagata and CMP resulted in complete protection against a PR8 (H1N1) challenge in A/Yamagata (H1N1)-vaccinated mice, while that with A/Guizhou (H3N2) and CMP exhibited a 100-fold reduction of nasal virus titer, demonstrating the cross-protective effect of CMP and influenza vaccine. It is suggested that CMP provide a safe and effective adjuvant for nasal vaccination with inactivated influenza vaccine.     

Enzyme immunoassay, complement fixation and hemagglutination inhibition tests in the diagnosis of influenza A and B virus infections. Purified hemagglutinin in subtype-specific diagnosis

The efficacy of enzyme immunoassay (EIA) in detecting diagnostic antibody rises to influenza A and B viruses was compared with complement fixation (CF) and hemagglutination inhibition (HI) tests in 455 patients with an acute respiratory infection. EIA and HI detected significantly more diagnostic antibody rises against influenza A than the CF method (96 and 87 vs. 47, respectively). In the case of influenza B significantly more diagnostic influenza B antibody rises were observed by EIA than by CF or HI (59 vs. 37 and 40, respectively). In most of the cases antibody rises in EIA were found in both IgG and IgA isotypes whereas increases in IgM antibodies were seen less frequently. Purified hemagglutinins (HA) were prepared from influenza A HI- and H3-subtypes and from influenza B viruses and used as antigens in EIA and the results were compared with those of HI. Infections caused by influenza A HI-subtype showed good homologous antibody responses in EIA but heterologous antibody responses to H3-subtype and influenza B HAs were frequently observed. Heterologous responses were clearly less frequent in patients with infections caused by the H3-subtype. Influenza B infections occasionally raised HA antibodies against influenza A H1-subtype but not to the H3-subtype. Interestingly, HI detected these heterologous responses at least as frequently as EIA. When whole viruses were used as antigens in EIA, subtype specificity was not observed and cross-reactions between influenza A and B virus antibodies were found. These observations suggest that, although EIA can show greater diagnostic efficacy over HI and CF methods, HI is still the serological method of choice in determining the causative subtype of influenza A virus infection.     

Comparison of long-term systemic and secretory antibody responses in children given live, attenuated, or inactivated influenza A vaccine

A comparison of inactivated intramuscular and live intranasal influenza A vaccines in young children undergoing primary immunization might be expected to show differences in serum and local mucosal antibody responses. To demonstrate such differences, serum and local respiratory tract antibody responses of young children vaccinated with intranasal live, attenuated, cold-adapted (H3N2 or H1N1), or intramuscular inactivated (H3N2) influenza A vaccines were examined for one year after vaccination. Antibody responses were measured by hemagglutination-inhibition (HAI) and class-specific enzyme-linked immunosorbent assay (ELISA). One year after vaccination, live intranasal vaccinees had significantly less decay of serum HAI (p = 0.025) and IgG antibody (p = 0.01) directed against the influenza hemagglutinin and neuraminidase than did intramuscular inactivated vaccinees. Nasal secretory IgA developed almost exclusively in live vaccinees and persisted for up to one year. Persistent nasal secretory IgG was detected in both live and inactivated vaccinees. Live vaccination not only stimulates a more durable serum antibody response, but also induces long-lasting local respiratory tract IgA antibody that may play an important role in host protection.     

Intranasal immunization with live attenuated influenza vaccine plus chitosan as an adjuvant protects mice against homologous and heterologous virus challenge

Our previous studies have proven the adjuvanticity of chitosan in mice when administered with inactivated and subunit influenza vaccine. In this study, we investigated the adjuvant effect of chitosan on the immunogenicity and protective efficacy of a live attenuated influenza vaccine. Mice were inoculated intranasally with live attenuated influenza vaccine plus chitosan and then challenged with a high, lethal dose of homologous or heterologous virus. Antibody responses, secretion of IFN-γ by spleen cells, body weight loss, survival rates, and residual lung virus titers were tested. The results demonstrated that live attenuated influenza vaccine with chitosan adjuvant not only protected mice completely against challenge with the homologous virus but also provided good cross-protection against a heterologous virus. In addition, chitosan as adjuvant could significantly increase the levels of antigen-specific antibodies and the population of IFN-γ-secreting T cells. These results reveal the potential of chitosan as a candidate adjuvant for use in a live attenuated influenza vaccine.     

Immunoglobulin A-deficient mice exhibit altered T helper 1-type immune responses but retain mucosal immunity to influenza virus

We have previously demonstrated that immunoglobulin A (IgA)(-/-) knockout (KO) mice exhibit levels of susceptibility to influenza virus infection that are similar to those of their normal IgA(+/+) littermates. To understand the mechanism of this apparent mucosal immunity without IgA, immunoglobulin isotype and T helper 1 (Th1)-type [interferon-gamma (IFN-gamma)] and Th2-type [interleukin (IL)-4, IL-5)] cytokine responses to influenza vaccine were evaluated. Intranasal immunization with influenza virus subunit vaccine plus cholera toxin/cholera toxin B subunit (CT/CTB) induced significant influenza virus-specific immunoglobulin G (IgG) antibody in the serum and nasal passages of both IgA(-/-) and IgA(+/+) mice, while IgA antibodies were induced only in IgA(+/+) mice. IgA KO mice exhibited an IgG1 subclass haemagglutinin (HA)-specific response but no detectable IgG2a and IgG2b responses. In contrast, IgA(+/+) mice exhibited significant IgG1 as well as IgG2a responses. This indicates a predominant Th2-type response in IgA KO mice compared to normal mice. Following stimulation with influenza virus in vitro, splenic lymphocytes from immunized IgA(-/-) mice produced significantly lower levels of IFN-gamma than IgA(+/+) mice (P < 0.001), but elaborated similar levels of IL-4 and IL-5. This was true at both protein and mRNA levels. Immunized mice were challenged intranasally with a small inoculum of influenza virus to allow deposition of virus in the nasal mucosal passages. Compared to non-immunized mice, immunized IgA(-/-) and IgA(+/+) mice exhibited significant, but similar levels of reduction in virus titres in the nose and lung. These results demonstrate that in addition to IgA deficiency, IgA gene deletion also resulted in down-regulated Th1-type immune responses and confirm our previous data that IgA antibody is not indispensable for the prevention of influenza virus infection.     

Secretory IgA antibodies provide cross-protection against infection with different strains of influenza B virus

This study examined whether secretory IgA (S-IgA) antibodies (Abs) could confer cross-protective immunity against infection with influenza B viruses of antigenically distinct lineages. Wild-type or polymeric Ig receptor (pIgR)-knockout (KO) mice were immunized by infection with different B viruses or by intranasal (i.n.) administration with different inactivated vaccines. Four weeks later mice were challenged with either the B/Ibaraki/2/85 virus, representative of the B/Victoria/2/87 (B/Victoria)-lineage, or B/Yamagata/16/88 virus, representative of the B/Yamagata-lineage. Three days after challenge, nasal wash and serum specimens were assayed for IgA and IgG Abs specific for challenge viral antigens and for protection against challenge viruses. In wild-type mice, B/Ibaraki (or B/Yamagata) cross-reactive IgA Abs were detected at higher levels when infected or immunized with homologous-lineage viruses and at lower levels when infected or immunized with heterologous-lineage viruses. There was a correlation between the amount of nasal cross-reactive IgA Ab and the efficacy of cross-protection with a homologous-lineage virus. In mice lacking the pIgR, nasal cross-protective IgA Abs were only marginally detected in vaccinated mice and an accumulation of IgA in the serum was observed. This reduction of nasal IgA was accompanied by inefficient cross-protection against the B/Ibaraki (or B/Yamagata) virus infection. These results suggest that challenge viral-antigen cross-reactive S-IgA in nasal secretions induced by i.n. infection or vaccination is involved in providing cross-protection against challenge infection with virus within either the B/Victoria- or B/Yamagata-lineage.     

Vaccination with CpG-adjuvanted avian influenza virosomes promotes antiviral immune responses and reduces virus shedding in chickens

The use of virosomes as a vaccine platform has proven successful against several viruses. Here we examined the protective efficacy of a virosome-based vaccine consisting of avian influenza virus (AIV) A/Duck/Czech/56/H4N6 in chickens against a homologous AIV challenge. Virosomes adjuvanted with CpG-ODN or recombinant chicken interferon (IFN)-γ significantly reduced virus shedding after virus challenge. Furthermore, immunization with virosomes adjuvanted with CpG-ODN increased hemagglutination inhibition (HI) and virus-specific neutralizing serum antibodies, as well as virus-specific serum IgG and mucosal IgA responses. We also found a significant increase in the expression of type I and II interferon genes in the protected birds following virus challenge. In summary, this study demonstrated the ability of virosomes adjuvanted with CpG-ODN to reduce AIV shedding, and elicit virus-specific protective antibody responses in vaccinated birds.     

Intranasal immunisation with inactivated RSV and bacterial adjuvants induces mucosal protection and abrogates eosinophilia upon challenge

We have previously shown that following intranasal exposure to influenza virus, specific plasma cells are generated in the nasal-associated lymphoid tissue (NALT) and maintained for the life of the animal. However, we also showed that following infection with respiratory syncytial virus (RSV), specific plasma cells are generated in the NALT but wane quickly and are not maintained even after challenge, even though RSV-specific serum antibody responses remain robust. Only infection with influenza virus generated sterilising immunity, implying a role for these long-lived plasma cells in protection. We show here that the RSV-specific IgA NALT plasma cell population and lung antibody levels can be substantially boosted, both at acute and memory time points, by intranasal immunisation with inactivated RSV (iRSV) in combination with bacterial outer membrane vesicles (OMV) compared to live RSV alone. Finally, challenge with live RSV showed that immunisation with iRSV and OMV protect against both virus replication in the lung and the eosinophil infiltrate generated by either live RSV or iRSV alone. These data show that immunisation with iRSV and OMV maintains a NALT RSV-specific plasma cell population and generates an efficient protective immune response following RSV infection.     

IgM,IgG, and IgA Antibody Responses to Influenza A(H1N1)pdm09 Hemagglutinin in Infected Persons during the First Wave of the 2009 Pandemic in the United States

The novel influenza A(H1N1)pdm09 virus caused an influenza pandemic in 2009. IgM, IgG, and IgA antibody responses to A(H1N1)pdm09 hemagglutinin (HA) following A(H1N1)pdm09 virus infection were analyzed to understand antibody isotype responses. Age-matched control sera collected from U.S. residents in 2007 and 2008 were used to establish baseline levels of cross-reactive antibodies. IgM responses often used as indicators of primary virus infection were mainly detected in young patient groups (≤5 years and 6 to 15 years old), not in older age groups, despite the genetic and antigenic differences between the HA of A(H1N1)pdm09 virus and pre-2009 seasonal H1N1 viruses. IgG and IgA responses to A(H1N1)pdm09 HA were detected in all age groups of infected persons. In persons 17 to 80 years old, paired acute- and convalescent-phase serum samples demonstrated ≥4-fold increases in the IgG and IgA responses to A(H1N1)pdm09 HA in 80% and 67% of A(H1N1)pdm09 virus-infected persons, respectively. The IgG antibody response to A(H1N1)pdm09 HA was cross-reactive with HAs from H1, H3, H5, and H13 subtypes, suggesting that infections with subtypes other than A(H1N1)pdm09 might result in false positives by enzyme-linked immunosorbent assay (ELISA). Lower sensitivity compared to hemagglutination inhibition and microneutralization assays and the detection of cross-reactive antibodies against homologous and heterologous subtype are major drawbacks for the application of ELISA in influenza serologic studies.     

Superior cross-protective effect of nasal vaccination to subcutaneous inoculation with influenza hemagglutinin vaccine.

Intranasal (i.n.) vs. subcutaneous (s.c.) administration of influenza hemagglutinin (HA) vaccine was systematically compared in BALB/c mice. Mice were immunized with different vaccines, together with cholera toxin B subunit as an adjuvant, and 4 weeks later were challenged with either a small (2 microliters) or a large (20 microliters) volume of mouse-adapted A/Guizhou-X (H3N2) virus, each of which gave virgin mice either a nasal or a lung predominant infection. Both i.n. and s.c. inoculations of A/Guizhou-X vaccine conferred almost complete protection against both challenges, i.n. inoculation of A/Fukuoka (H3N2) or A/Sichuan (H3N2) vaccine conferred almost complete cross-protection against 2-microliters challenge and a partial cross-protection against 20-microliters challenge, whereas the s.c. inoculation conferred no cross-protection against 2-microliters challenge with a partial cross-protection against 20-microliters challenge. Moreover, i.n. immunization of PR8 (H1N1) vaccine gave a slight cross-protection against 2-microliters challenge, while the s.c. inoculation did not. The degree of protection was easily improved by i.n. inoculation of higher doses of vaccine, but not by the s.c. inoculation. In parallel with the protection, the i.n. vaccination produced a high level of cross-reacting IgA and IgG antibody to A/Guizhou-X HA in nasal and broncho-alveolar washes, while the s.c. vaccination produced the cross-reacting IgG antibody alone. Thus, i.n. inoculation with inactivated vaccines, which induces cross-reacting anti-HA IgA antibody as well as IgG antibody, is more effective than s.c. vaccination for providing cross-protection against drift viruses.