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J Virol. 2013 January; 87(2): 1261–1263.
PMCID: PMC3554089

Seasonal Trivalent Inactivated Influenza Vaccine Does Not Protect against Newly Emerging Variants of Influenza A (H3N2v) Virus in Ferrets


The recent increase in human cases of influenza A H3N2 variant virus [A(H3N2)v] highlights the need to assess whether seasonal influenza vaccination provides cross-protection against A(H3N2)v virus. Our data demonstrate that the 2011-2012 trivalent inactivated influenza vaccine (TIV) protected ferrets against homologous H3N2 virus challenge but provided minimal to no protection against A(H3N2)v virus. The complete absence of specific hemagglutination inhibition antibody response to A(H3N2)v is consistent with the poor cross-protection observed among TIV-immune animals.


Influenza A viruses have been isolated from swine since 1930 (1) and have been known to spread and cause disease in this species since the 1918 pandemic (2). The classical H1N1 virus was the predominant subtype isolated from U.S. swine until the late 1990s, when human H3N2 viruses infected this species and subsequently spread widely in North American pigs (3). Since that time, multiple reassortment events that have presumably occurred in swine have resulted in the emergence of an H3N2 virus with a triple reassortant internal gene (TRIG) cassette (4). The TRIG cassette, which shares host gene lineage origins with the A(H1N1)pdm09 virus, highlights the public health threat posed by swine-origin influenza subtypes (5).

Until recently, transmission of novel variants of H3N2 [A(H3N2)v] from swine to humans was rare, with only 7 confirmed cases documented in 2009-2010 (68). In 2011, public health laboratories in the United States detected an additional 12 cases of human infection (9, 10), caused by a novel A(H3N2)v virus that had acquired the M gene from A(H1N1)pdm09 virus (7). Since July 2012, there has been a substantial increase of swine-to-human transmission of A(H3N2)v virus (11). As of 19 October 2012, there have been 307 additional confirmed cases (including hospitalizations) among 11 U.S. states (12). Clinical characteristics of the A(H3N2)v cases have been generally consistent with signs and symptoms of seasonal influenza, and there is no evidence at this time that sustained human-to-human transmission is occurring. However, rare instances of probable human-to-human transmission associated with A(H3N2)v cases have occurred, and findings from an experimental study suggest that A(H3N2)v viruses have the capacity for efficient replication and transmission in mammals (13).

Vaccination is the most effective measure to control influenza. The seasonal H3N2 vaccine component present in the 2010-2011 and 2011-2012 trivalent inactivated influenza vaccine (TIV) is A/Perth/16/2009 (Perth/16; H3N2)-like viruses (14). Although serological studies indicate that Perth/16 (H3N2) and A(H3N2)v viruses are antigenically distinct from each other (7, 8), the efficacy of seasonal influenza vaccination against A(H3N2)v has not been adequately evaluated in vivo.

In this study, we evaluated whether the 2011-2012 TIV protected ferrets against A(H3N2)v A/Indiana/08/2011 (IN/11) virus challenge. Male Fitch ferrets (Triple F Farms, Sayre, PA), 8 to 12 months of age and seronegative against currently circulating human influenza H1, H3, and type B viruses, were vaccinated and twice boosted (3 to 4 weeks between injections) intramuscularly with an adult human dose (0.5 ml) of the 2011-2012 seasonal inactivated split-product TIV or phosphate-buffered saline (PBS) (controls) (15). Prior to vaccine boost and viral challenge, ferret sera were collected to assess hemagglutination inhibition (HI) antibody responses against IN/11 virus and the three homologous viruses in the 2011-2012 TIV. As shown in Table 1, all TIV-vaccinated ferrets displayed HI antibody titers of ≥80 against all three homologous viruses present in the TIV; however, cross-reactive HI antibodies to A(H3N2)v IN/11 virus were not observed.

Table 1
Serum hemagglutination inhibition antibody responses to TIV immunization in ferrets

We first determined the level of protection, induced by seasonal TIV against seasonal homologous Perth/16 (H3N2) virus challenge. The Perth/16 virus stock was grown in the allantoic cavities of 10-day-old embryonated hens' eggs at 34°C for 48 h and titrated in a standard plaque assay expressed as PFU. Ferrets were challenged intranasally with 106 PFU of virus, and vaccine protection was measured by reduction in fever, weight loss, and upper respiratory tract virus replication (15). Viral challenge with the seasonal Perth/16 virus resulted in minimal morbidity among vaccinated and control ferrets, causing 3.4% and 3.9% maximum weight loss, respectively (Table 2). No significant differences in body temperatures were detected between TIV-immune and unimmunized control ferrets observed for 14 days postchallenge (p.c.), although there was a trend toward reduced fevers among TIV-immune animals. The extent of virus replication in the upper respiratory tract was determined by titrating nasal wash samples collected on alternating days p.c. The TIV did not provide sterilizing immunity against homologous viral challenge, as seen previously (15), and viral titers were observed in all TIV-immunized ferrets and control ferrets (Fig. 1). However, in comparison to control ferrets, TIV-immunized ferrets displayed a significant reduction in viral titers on every day analyzed (day 2, P = 0.007; day 4, P = 0.03; and day 6, P = 0.04), until viral clearance was observed in both groups on day 8 p.c.

Table 2
Clinical symptoms observed in TIV-immune ferrets challenged with homologous or A(H3N2)v virus
Fig 1
TIV vaccine efficacy following challenge with A/Perth/16/2009 (Perth/16) or A/Indiana/08/11 (IN/11) virus. Nasal washes were collected with 1 ml of PBS on even-numbered days postchallenge. Titers were determined by standard plaque assay on MDCK cells ...

Next, we assessed the degree of cross-protection against the A(H3N2)v IN/11 virus conferred by seasonal TIV. Ferrets were challenged intranasally with 106 PFU of the IN/11 stock virus, which was grown in Madin-Darby canine kidney (MDCK) cells. Overall, ferrets challenged with IN/11 virus displayed higher temperatures and greater weight loss than ferrets challenged with Perth/16 virus (Table 2). On day 2 p.c., all unimmunized control ferrets exhibited an early spike in body temperature, ranging from 0.5 to 1.8°C over baseline (mean maximum = 1.2°C) (Table 2). Similarly, TIV-immune animals also displayed an early spike in body temperature, ranging from 0.75 to 1.8°C over baseline (mean maximum = 1.5°C). Moreover, in comparison to control animals, TIV-immune ferrets did not display significant differences in weight loss and virus titers on peak days (2 to 6 days p.c.) of replication (Table 2 and Fig. 1). However, TIV-immune ferrets showed a modest reduction in viral titers on day 8 (P = 0.02), perhaps due to a low level of anti-N2 neuraminidase cross-reactive antibodies induced by the TIV (16).

The results of this study suggest that previous immunization with the commercially available seasonal TIV may provide minimal to no cross-protection against A(H3N2)v virus. These data are consistent with human serologic studies demonstrating that immunization with the 2010-2011 TIV has no impact on the level of cross-reactive A(H3N2)v antibodies in immunologically naive children (age, <3 years) and failed to substantially improve the level of cross-reactive antibodies in adults (17, 18). Because the majority of the population lacks specific immunity against this new virus variant, an A(H3N2)v-specific vaccine is needed for optimal protection for all ages.


K.V.H. received financial support for this work from the Oak Ridge Institute for Science and Education, Oak Ridge, TN.

The findings and conclusions in this report are those of the authors and do not necessarily reflect the views of the funding agency.


Published ahead of print 31 October 2012


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