Immunity to pre-1950 H1N1 influenza viruses confers cross-protection against the pandemic swine-origin 2009 A (H1N1) influenza virus

Abstract

The 2009 H1N1 influenza virus outbreak is the first pandemic of the twenty-first century. Epidemiological data reveal that of all the people afflicted with H1N1 virus, <5% are over 51 y of age. Interestingly, in the uninfected population, 33% of those >60 y old have pre-existing neutralizing Abs against the 2009 H1N1 virus. This finding suggests that influenza strains that circulated 50-60 y ago might provide cross-protection against the swine-origin 2009 H1N1 influenza virus. To test this, we determined the ability of representative H1N1 influenza viruses that circulated in the human population from 1930 to 2000, to induce cross-reactivity to and cross-protection against the pandemic swine-origin H1N1 virus, A/California/04/09. We show that exposure of mice to the 1947 virus, A/FM/1/47, or the 1934 virus, A/PR/8/34, induced robust cross-protective immune responses and these mice were protected against a lethal challenge with mouse-adapted A/California/04/09 H1N1 virus. Conversely, we observed that mice exposed to the 2009 H1N1 virus were protected against a lethal challenge with mouse-adapted 1947 or 1934 H1N1 viruses. In addition, exposure to the 2009 H1N1 virus induced broad cross-reactivity against H1N1 as well as H3N2 influenza viruses. Finally, we show that vaccination with the older H1N1 viruses, particularly A/FM/1/47, confers protective immunity against the 2009 pandemic H1N1 virus. Taken together, our data provide an explanation for the decreased susceptibility of the elderly to the 2009 H1N1 outbreak and demonstrate that vaccination with the pre-1950 influenza strains can cross-protect against the pandemic swine-origin 2009 H1N1 influenza virus.

FIGURE 1

Sera from mice sublethally infected with pre-1950 influenza strains cross-react with the swine-origin H1N1 influenza virus, A/CA/04/2009. Cohorts of BALB/c mice were infected intranasally with 0.1 × LD₅₀ of live mouse-adapted influenza viruses spanning from 1930 to 2000 (A/PR/8/34, A/FM/1/47, A/Denver/1/57, A/California/10/78, A/Chile/1/83, and A/New Caledonia/20/99). A month following infection, we collected their sera and tested their ability to inhibit hemagglutination of turkey RBCs by the 2009 swine-origin A/California/04/09 virus. The HAI titers were read as the reciprocal of the highest dilution of serum that conferred inhibition of hemagglutination. The values are expressed as mean ± SD. Each data point represents an individual animal.

FIGURE 2

Exposure to A/PR/8/34 and A/FM/1/47 induces protective immunity against A/California/04/09. Cohorts of BALB/c mice were sublethally infected intranasally with 0.1 × LD₅₀ live mouse-adapted A/PR/8/34 and A/FM/1/47 influenza viruses. We collected sera from these mice at days 7, 14, and 28 and determined their A/California/04/09-specific IgG titers (A) and HAI titers (B). One month later, these mice were challenged with 3 × LD₅₀ live mouse-adapted A/California/04/09 virus and monitored for survival up to 14 d postchallenge (C). Body weight changes, indicative of morbidity, were also monitored up to 14 d (D). The graphs represent the mean ± SEM.

FIGURE 3

Abs produced in response to A/California/04/09 cross-react with a large spectrum of older H1N1 influenza viruses. We infected cohorts of BALB/c/mice sublethally with live mouse-adapted swine-origin A/CA/04/2009, collected their sera 1 mo postinfection, and tested their ability to cross-react with a panel of 1930–2000 H1N1 viruses, using HAI assay (A). We then challenged these animals with 3 × LD₅₀ mouse-adapted A/PR/8/34 or A/FM/1/47 strains and monitored for survival (B) and signs of morbidity (C), as reflected in the body weight changes up to 14 d postchallenge. The graphs represent the mean ± SD.

FIGURE 4

Exposure to A/California/04/09 induces heterosubtypic cross-reactivity and protective immunity against H3N2 influenza viruses. We infected cohorts of BALB/c/mice sublethally with live mouse-adapted swine-origin A/CA/04/2009, collected their sera 1 mo postinfection, and tested their ability to cross-react with a panel of H3N2 viruses, using HAI assay (A). We then challenged these animals with 3 × LD₅₀ mouse-adapted H3N2 viruses, A/Aichi/2/68 or A/Victoria/3/75, and monitored them for survival (B) and signs of morbidity (C), as reflected in the body weight changes up to 14 d postchallenge. The graphs represent the mean ± SD of five mice per group.

FIGURE 5

Vaccination with inactivated A/FM/1/47 or A/PR/8/34 virus induces robust Ab responses and protective immunity against A/CA/04/2009. We immunized cohorts of BALB/c mice with 10 μg (1800 HA units) of formalin-inactivated A/FM/1/47 or A/PR/8/34 influenza virus. We collected their sera at days 7, 14, and 28 postimmunization and determined the A/California/04/09-specific ELISA (A) and HAI titers (B). Naive, unimmunized mice served as negative controls. The results are the average of two independent experiments, and error bars represent SEM. One month following immunization, we challenged them by intranasal infection with 3 × LD₅₀ of live mouse-adapted A/California/04/09 virus. Postchallenge, the survival rates (C) and body weight changes (D), indicative of morbidity, were monitored for 14 d. The graphs represent the mean ± SEM of five mice per group at each time point.

FIGURE 6

CD8 T cells are critical for cross-protection. Cohorts of BALB/c mice sublethally infected with 0.1 × LD₅₀ live mouse-adapted swine origin A/CA/04/2009 and allowed to proceed to memory phase were depleted of CD8 T cells with anti-CD8 mAbs. Following CD8 depletion, these mice were challenged intranasally with 3 × LD₅₀ mouse-adapted A/FM/1/47 or A/Aichi/2/68 strains and monitored for survival (A) and body weight changes (B) up to 14 d postchallenge. Immune yet non–CD8-depleted mice and naive mice served as controls. The body weight changes of the survivors are plotted as mean ± SEM.

FIGURE 7

A, Phylogenetic analysis of the 1930–2000 H1N1 influenza virus strains. The H1 HA amino acid sequence from A/California/04/09 was aligned to the HA amino acid sequences from A/Puerto Rico/8/34, A/Fort Monmouth/1/47, A/Denver/1/57, A/California/10/1978, A/Chile/1/83, and A/New Caledonia/20/99. The multisequence alignment and the phylogenetic analysis showed the sequence identity of the tested isolates to the swine-origin H1N1 and their relatedness. Comparative modeling of A/PR/8/34, A/FM/1/47, and A/California/04/09 are shown in B–G. The HA proteins were superimposed onto the crystal structure of the A/PR/8/34 HA trimer. The differences in primary amino acid sequence between the A/California/04/09 HA and the A/FM/1/47 (B, D, F) and the A/California/04/09 HA and A/PR/8/34 (C, E, G) are highlighted in yellow. Antigenic sites determined by mapping on the A/PR/8/34 HA protein (22) are highlighted in cyan, and regions of overlap are highlighted in green.