Research Article: Protection of Mice against Lethal Challenge with 2009 H1N1 Influenza A Virus by 1918-Like and Classical Swine H1N1 Based Vaccines

Date Published: January 29, 2010

Publisher: Public Library of Science

Author(s): Balaji Manicassamy, Rafael A. Medina, Rong Hai, Tshidi Tsibane, Silke Stertz, Estanislao Nistal-Villán, Peter Palese, Christopher F. Basler, Adolfo García-Sastre, Ron A. M. Fouchier.


The recent 2009 pandemic H1N1 virus infection in humans has resulted in nearly 5,000 deaths worldwide. Early epidemiological findings indicated a low level of infection in the older population (>65 years) with the pandemic virus, and a greater susceptibility in people younger than 35 years of age, a phenomenon correlated with the presence of cross-reactive immunity in the older population. It is unclear what virus(es) might be responsible for this apparent cross-protection against the 2009 pandemic H1N1 virus. We describe a mouse lethal challenge model for the 2009 pandemic H1N1 strain, used together with a panel of inactivated H1N1 virus vaccines and hemagglutinin (HA) monoclonal antibodies to dissect the possible humoral antigenic determinants of pre-existing immunity against this virus in the human population. By hemagglutinination inhibition (HI) assays and vaccination/challenge studies, we demonstrate that the 2009 pandemic H1N1 virus is antigenically similar to human H1N1 viruses that circulated from 1918–1943 and to classical swine H1N1 viruses. Antibodies elicited against 1918-like or classical swine H1N1 vaccines completely protect C57B/6 mice from lethal challenge with the influenza A/Netherlands/602/2009 virus isolate. In contrast, contemporary H1N1 vaccines afforded only partial protection. Passive immunization with cross-reactive monoclonal antibodies (mAbs) raised against either 1918 or A/California/04/2009 HA proteins offered full protection from death. Analysis of mAb antibody escape mutants, generated by selection of 2009 H1N1 virus with these mAbs, indicate that antigenic site Sa is one of the conserved cross-protective epitopes. Our findings in mice agree with serological data showing high prevalence of 2009 H1N1 cross-reactive antibodies only in the older population, indicating that prior infection with 1918-like viruses or vaccination against the 1976 swine H1N1 virus in the USA are likely to provide protection against the 2009 pandemic H1N1 virus. This data provides a mechanistic basis for the protection seen in the older population, and emphasizes a rationale for including vaccination of the younger, naïve population. Our results also support the notion that pigs can act as an animal reservoir where influenza virus HAs become antigenically frozen for long periods of time, facilitating the generation of human pandemic viruses.

Partial Text

Influenza A viruses (IAV), members of the Orthomyxoviridae family, cause severe respiratory diseases in humans with an average mortality rate of 36,000/year in the United States alone [1]. Apart from yearly seasonal outbreaks, IAV can cause frequent epidemics and occasional pandemics in humans [2],[3]. Vaccination has been one of the most effective means of protection against IAV. Vaccine induced production of antibodies against the viral surface glycoprotein hemagglutinin (HA) is crucial for immune protection [4]. The HA plays a critical role in the virus life cycle by mediating virus binding to sialic acid containing receptors on the cell surface and fusion of viral and endosomal membranes, leading to viral entry into the host cell [2],[5]. HA-specific antibodies have been demonstrated to block the IAV infection by preventing receptor binding and/or fusion. However, the HA protein, due to antibody mediated immune selection pressure, undergoes rapid antigenic evolution by accumulation of mutations (“antigenic drift”) and through genetic reassortments of segments (“antigenic shift”). In the 20th century, influenza virus caused three pandemics in humans: 1918 “Spanish influenza” (H1N1), 1957 “Asian influenza” (H2N2) and 1968 “Hong Kong influenza” (H3N2) [6],[7]. In April 2009 the Centers for Disease Control and Prevention (CDC) of the United States of America announced the detection of a novel strain of influenza virus in humans. Further investigation revealed that this novel virus derived its genes from viruses circulating in the pig population. Due to sustained human-to-human transmission of this novel virus throughout the world on June 11th, the World Health Organization (WHO) raised the worldwide pandemic alert level to phase 6 (e.g. ongoing global spread and community level outbreaks in multiple parts of world). All of the past pandemics and the recent 2009 swine-origin IAV H1N1 pandemic have been caused by IAV strains carrying an antigenically novel HA segment in populations immunologically naïve to that particular HA.

Seasonal influenza viruses predominantly cause severe disease in very young children and in the older population. However, infections with the 2009 pandemic H1N1 are considerably lower in people 65 years or older, likely due to immunity from prior exposure and/or vaccination to an antigenically similar influenza virus [24],[25],[26]. Using a panel of 11 different influenza viruses from 1918–2009, we tested the ability of inactivated vaccines based on these viruses to protect against a mouse-lethal 2009 pandemic H1N1. Here, we show that vaccination of mice with human 1918 influenza virus VLPs, and inactivated human Wei/43 and classical swine H1N1 viruses completely protects from death by lethal challenge with the 2009 pandemic H1N1. Also, analysis of pre-challenge sera from mice vaccinated with these viruses, except Wei/43, show cross-reactivity against the 2009 H1N1 virus (HI titer ≥40). In contrast, vaccination with more contemporary H1N1 viruses offered only partial protection. In addition, passive immunization with 1918 HA-specific monoclonal antibodies protects against the 2009 H1N1 virus, suggesting antigenic similarity between these viruses. Cross-protective epitope mapping shows that 1918 HA-specific mAbs protect by binding to the conserved antigenic site Sa in 2009 H1N1 virus. Based on our HI data (Table 2) and since inactivated vaccines are known to induce protective humoral and minimal cellular immunity, this protection was likely mediated by antibodies, as also evidenced by the lack of complete protection by H3N2-based vaccines. Our results indicate that prior exposure to human influenza A viruses from 1918–1943 or vaccination with classical swine H1N1 virus (NJ/76) offers significant levels of cross-protection against the novel 2009 H1N1 pandemic virus and thus provides a mechanistic explanation for the lower incidence of disease and/or infection seen in people aged 65 or older.