Vaccine-induced NA immunity decreases viral shedding, but does not disrupt chains of airborne transmission for the 2009 pandemic H1N1 virus in ferrets

Abstract

Split-virion-inactivated influenza vaccines are formulated based on viral hemagglutinin content. These vaccines also contain the viral neuraminidase (NA) protein, but NA content is not standardized and varies between manufacturers. In clinical studies and animal models, antibodies directed toward NA reduced disease severity and viral load; however, the impact of vaccine-induced NA immunity on airborne transmission of influenza A viruses is not well characterized. Therefore, we evaluated if vaccination against NA could disrupt chains of airborne transmission for the 2009 pandemic H1N1 virus in ferrets. Immunologically naïve donor ferrets were infected with the 2009 pandemic H1N1 virus and then paired in transmission cages with mock- or NA-vaccinated respiratory contacts. The mock- and NA-vaccinated animals were then monitored daily for infection, and once infected, these animals were paired with a naive secondary respiratory contact. In these studies, all mock- and NA-vaccinated animals became infected; however, NA-vaccinated animals shed significantly less virus for fewer days relative to mock-vaccinated animals. For the secondary contacts, 6/6 and 5/6 animals became infected after exposure to mock- and NA-vaccinated animals, respectively. To determine if vaccine-induced immune pressure selected for escape variants, we sequenced viruses recovered from ferrets. No mutations in NA became enriched during transmission. These findings indicate that despite reducing viral load, vaccine-induced NA immunity does not prevent infection during continuous airborne exposure and subsequent onward airborne transmission of the 2009 pandemic H1N1 virus.

Importance: In humans and animal models, immunity against neuraminidase (NA) reduces disease severity and viral replication during influenza infection. However, we have a limited understanding of the impact of NA immunity on viral transmission. Using chains of airborne transmission in ferrets as a strategy to simulate a more natural route of infection, we assessed if vaccine-induced NA immunity could disrupt transmission of the 2009 pandemic H1N1 virus. The 2009 pandemic H1N1 virus transmitted efficiently through chains of transmission in the presence of NA immunity, but NA-vaccinated animals shed significantly less virus and had accelerated viral clearance. To determine if immune pressure led to the generation of escape variants, viruses in ferret nasal wash samples were sequenced, and no mutations in NA were identified. These findings demonstrate that vaccine-induced NA immunity is not sufficient to prevent infection via airborne exposure and onward airborne transmission of the 2009 pandemic H1N1 virus.

Keywords: airborne transmission; ferret model; influenza; influenza vaccines; neuraminidase.

Fig 1

Assessment of NA binding and activity inhibiting antibodies. Ferrets (n = 6/group) were given a mock or recombinant NA vaccine on days 0, 28, and 56, and blood samples were collected at regular intervals to assess the antibody response to NA. Panels (A) and (B) display NA-binding IgG antibody titers determined by ELISA, and NA activity inhibiting antibody titers determined by ELLA, respectively. In a separate study (31), ferrets were infected with the 2009 pandemic H1N1 virus, and blood samples were collected for 90 days. Panels (C) and (D) show NA binding IgG antibody titers and NA activity inhibiting antibody titers in ferrets over 90 days post-infection.

Fig 2

Sequential transmission of the 2009 pandemic H1N1 virus using mock-vaccinated ferrets as the RC1. Six ferrets designated as directly inoculated (DI) were intranasally infected with 1 × 10⁶ TCID₅₀ of recombinant A/California/07/2009 (H1N1pdm09) virus. Twenty-four hours later, each DI animal was introduced into a transmission cage with a mock-vaccinated respiratory contact (Mock RC1) ferret. Nasal wash samples were collected daily from the Mock RC1 and assayed for evidence of viral infection by qRT-PCR. When the RC1 ferret became infected, it was housed with a new respiratory contact (RC2). Each panel (A and F) represents an independent replicate of DI, Mock RC1, and RC2 (i.e., ferret trio). Magenta, green, and blue lines represent the nasal wash titers for the DI, Mock RC1, and RC2 ferrets, respectively. The black arrow denotes the day the DI and Mock RC1 were paired, and the orange arrow and dashed line indicate when the Mock RC1 and RC2 ferrets were paired. Ct values for RC1 animals on the day that RC1 and RC2 animals were paired are provided above the orange arrows. Nasal wash samples were titrated on MDCK cells, and the results are expressed as log₁₀ TCID₅₀/mL of nasal wash. The dashed horizontal line denotes the limit of detection (0.5 Log TCID50/mL).

Fig 3

Sequential transmission of the 2009 pandemic H1N1 virus using NA-vaccinated ferrets as the RC1. Six ferrets designated as directly inoculated (DI) were intranasally infected with 1 × 10⁶ TCID₅₀ of recombinant A/California/07/2009 (H1N1pdm09) virus. Twenty-four hours later, each DI animal was introduced into a transmission cage with an NA-vaccinated respiratory contact (NA RC1) ferret. Nasal wash samples were collected daily from the NA RC1 and assayed for evidence of viral infection by qRT-PCR. When the NA RC1 ferret became infected, it was housed with a new respiratory contact (RC2). Each panel (A–F) represents an independent replicate of DI, NA RC1, and RC2 (i.e., ferret trio). Magenta, green, and blue lines represent the nasal wash titers for the DI, NA RC1, and RC2 ferrets, respectively. The black arrow denotes the day the DI and NA RC1 were paired, and the orange arrow and dashed line indicate when the NA RC1 and RC2 ferrets were paired. Ct values for RC1 animals on the day that RC1 and RC2 animals were paired are provided above the orange arrows. Nasal wash samples were titrated on MDCK cells, and the results are expressed as log₁₀ TCID₅₀/mL of nasal wash. The dashed horizontal line denotes the limit of detection (0.5 Log TCID50/mL).

Fig 4

Analyses of viral shedding kinetics for transmission chain experiments using mock- and NA-vaccinated RC1 ferrets. To determine if vaccine-induced NA immunity altered viral shedding kinetics several analyses were performed. Shown in (A) and (B) are peak nasal wash titers and total viral shedding assessed by area under the curve analysis for ferrets in each experimental group, respectively. To assess if NA immunity altered the progression of viral replication, in (C), viral shedding curves for Mock RC1 and NA RC1 ferrets were overlaid with day 0 being the first-day vRNA was detected in the nasal wash. Shown are average titers at each timepoint in each group. Panel (D) displays days post-contact that ferrets in Mock RC1, NA RC1, Mock RC2, and NA RC2 groups began shedding virus after respiratory contact exposure to an infected animal. *significantly different P < 0.05.

Fig 5

Influenza A single nucleotide variants (SNVs) in mock- and NA-vaccinated RC1 ferret nasal wash samples. (A) The number of unique non-synonymous (left) and synonymous (right) SNVs found in each protein (x-axis) across all mock-vaccinated RC1 ferret nasal wash samples. (B) Boxplots representing the median and interquartile range of the relative frequency of SNVs (y-axis) identified in the mock-vaccinated RC1 nasal wash samples. Outliers are depicted as individual points. (C) The number of unique non-synonymous (left) and synonymous (right) SNVs found in each protein (x-axis) across all NA-vaccinated RC1 ferret samples. (D) Boxplots representing the median and interquartile range of the relative frequency of SNVs (y-axis) identified in the NA-vaccinated RC1 nasal wash samples. Outliers are depicted as individual points.

Fig 6

Non-synonymous mutations in the HA and NA proteins identified in two or more ferrets during sequential transmission. The displayed mutations were selected based on the criteria of being non-synonymous and occurring at frequencies of 1% (0.01) or higher in at least two samples, one of which had to be an RC1 sample collection. The relative frequency (y-axis) of (A) HA SNVs and (B) NA SNVs across the days of the experiment (x-axis). For both plots, data are grouped by the six mock-vaccinated and six NA-vaccinated transmission chains (t1–t6, across) and variants (down). The S column heading denotes the frequency of a variant in the viral stock used for infection. The point shape depicts if the variant is ≥1% (0.01, circle) or <1% (0.01, “X”) in the sample. The color of each point and line indicates the ferret in each transmission pair. DI = directly infected, RC1 = respiratory contact 1, and RC2 = respiratory contact 2. If the mutation is not observed in any of the six transmission chains for a given vaccination group, the plots will be empty. HA mutations are based on numbering from the H1 start codon.