Cross-subtype immunity against avian influenza in persons recently vaccinated for influenza.(RESEARCH)

Avian influenza virus (H5N1) can be transmitted to humans, resulting in a severe or fatal disease. The aim of this study was to evaluate the immune cross-reactivity between human and avian influenza (H5N1) strains in healthy donors vaccinated for seasonal influenza A (H 1 N 1)/(H3N2). A small frequency of CD4 T cells specific for subtype H5N1 was detected in several persons at baseline, and seasonal vaccine administration enhanced the frequency of such reactive CD4 T cells. We also observed that seasonal vaccination is able to raise neutralizing immunity against influenza (H5N1) in a large number of donors. No correlation between influenza-specific CD4 T cells and humoral responses was observed. N1 may possibly be a target for both cellular and humoral cross-type immunity, but additional experiments are needed to clarify this point. These findings highlight the possibility of boosting cross-type cellular and humoral immunity against highly pathogenic avian influenza A virus subtype H5N1 by seasonal influenza vaccination.

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Influenza viruses are segmented, negative-sense RNA viruses belonging to the family Orthomyxoviridae. According to the antigenic differences in nucleoprotein and matrix proteins, 3 types of influenza viruses (A, B, and C) have been described. Influenza viruses A and B are associated with seasonal illness and death, whereas influenza virus C causes mild infections (1,2). Influenza A viruses are subtyped on the basis of the antigenic differences on external hemagglutinin (HA) and neuraminidase (NA) glycoproteins. Human type A influenza virus subtypes have been limited to H1, H2, and H3 and to N1 and N2 (3). Several HAs and NAs have been isolated from avian hosts; occasionally, they have been associated with human outbreaks (4,5).

Cytotoxic T lymphocytes play a central role in the clearance of primary influenza virus infection, peaking after 7-10 days; the peak in antibody titers occurs 4-7 weeks after primary infection (6-8). Neutralizing antibodies are completely protective against secondary challenges only with closely related strains, but they are ineffective against viruses with major antigenic divergence. For this reason, current influenza vaccines are prepared annually on the basis of World Health Organization forecasts on the most probable influenza A and B virus strains thought to be circulating in the next seasonal outbreak (5,7). By contrast, cellular responses to cross-reactive epitopes may provide a substantial degree of protection against serologically distinct viruses (9). The ability of influenza viruses to mutate and reassort their HA-NA genome segments between different animal species is a main concern because immunity generated by previous infections or vaccinations is unable to prevent infection by itself, although it may reduce virus replication and spread (8-10).

To date, 3 influenza subtypes have produced pandemic disease in humans: H1N1 in 1918, H2N2 in 1957, and H3N2 in 1968 (4,11,12). In 1997, during the avian influenza (H5N 1) outbreak in Hong Kong Special Administrative Region, People's Republic of China, a cross-reactive cellular immune response induced by influenza (H9N2) was able to protect chickens from influenza (H5N1) (13). Moreover, adults living in the United States who were never exposed to H5N1 subtype have shown cross-type cellular immunity to influenza A virus strains derived from swine and avian species (including the H5N1 subtype isolated in Hong Kong) (14). Thus, speculation that cross-reactive T cells may decrease illness and death by reducing the replication of the new influenza virus, even if elicited by a different strain, is reasonable.

Avian influenza A viruses of the H5N1 subtype are currently causing widespread infections in bird populations. Numerous instances of transmission to humans have been recently reported in Asia and Africa, with the infection resulting in severe disease or death (>50% fatality rate). Hence, the aim of the present study was to evaluate the immune cross-reactivity between human and avian influenza (H5N1) strains in healthy donors recently vaccinated for seasonal influenza A (H1N1/H3N2). Our data indicate that influenza vaccination may boost cross-subtype immunity against influenza (H5N1), involving cellular or humoral responses or both.

Study Design and Methods

Study Population

Healthcare workers wishing to receive seasonal influenza vaccination at the Spallanzani Institute (n = 42) were enrolled. The study was approved by the local Ethical Committee; all participants gave written informed consent. Baseline characteristics of the study population are reported in the Table. Blood samples were obtained before (t0) and 30 days after vaccination (t1). The vaccine formulation was Fluarix, an inactivated and purified split influenza vaccine (GlaxoSmithKline, Verona, Italy). The antigen composition and strains were A/California/7/2004-H3N2; A/New Caledonia/20/99-H1N1; and B/Shanghai/361/2002. Each 0.5-mL vaccine dose contains 15 [micro]g HA of each strain in phosphate-buffered saline and excipients. Vaccine was administered intramuscularly.

Cells, Viruses, and Antigens

Madin-Darby-canine kidney (MDCK) cells were maintained in Dulbecco modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS), and 2 mmol/L L-glutamine, at 37[degrees]C in a 5%…