Immunity to hemagglutinin and neuraminidase results in additive reductions in airborne transmission of influenza H1N1 virus in ferrets

Abstract

Currently, there is limited knowledge on the impact of immunity to hemagglutinin (HA) and/or neuraminidase (NA) on the transmission of influenza viruses. Therefore, using intramuscular vaccination, intranasal vaccination, or infection with reassortant viruses, we induced immunity to each antigen alone or both antigens combined in ferrets. We then assessed transmission of the 2009 pandemic H1N1 virus from these ferrets to naïve respiratory contacts. For all strategies used to induce immunity, combined immunity to HA and NA resulted in the largest reductions in transmission. Moreover, immunity to HA and NA conferred additive rather than synergistic reductions in transmission. No escape variants emerged in our transmission studies, and logistical regression showed that the probability of transmission was less than 50% when viral titers in donors were reduced to 10^1.5 and 10² median tissue culture infectious dose per ml on days 1 and 3 postinfection, respectively. These studies define the relationship between immunity to HA and NA on transmission and identify a threshold titer indicative of decreased transmission in ferrets.

Fig. 1.. Assessment of the antibody response in IM and IN vaccinated ferrets before 2009 H1N1 infection.

Serum samples collected on day 82 post–primary vaccination from ferrets (n = 3 or 4 per group) given mock, HA, NA, or HA + NA vaccines via IM or IN routes were analyzed for antibodies to H1 HA and N1 NA. (A) H1 and N1 IgG binding antibody titers, and (B) HAI, neutralization, and NAI antibody titers in IM vaccinated ferrets. (C) H1 and N1 IgG binding antibody titers, and (D) HAI, neutralization, and NAI titers in IN vaccinated ferrets. Binding antibody titers were determined by ELISA. HAI and neutralization titers were determined via HAI assay and microneutralization assays, respectively. NAI titers were determined via ELLA. ELISA, HAI, and ELLA assays were performed in duplicate, and microneutralization assays were performed with four technical replicates. For all antibody analyses, individual values are displayed with horizontal line, and error bars showing mean ± SEM, respectively. Orange triangles, dark blue circles, pink squares, and green inverted triangles represent mock-, HA-, NA-, and IM HA + NA–vaccinated DRs, respectively. ^*Significantly different from mock- and NA-vaccinated DRs (P < 0.02). ^†Significantly different from mock- and HA-vaccinated DRs (P < 0.02) determined via Kruskal-Wallis test with post hoc two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli.

Fig. 2.. Airborne transmission of the 2009 H1N1 virus from IM vaccinated preimmune DRs to immunologically naïve RC ferrets.

IM mock-, HA-, NA-, and HA + NA–vaccinated ferrets (n = 3 or 4 per group) were IN inoculated with 1 × 10⁴ TCID₅₀ of the 2009 H1N1 virus. Twenty-four hours later, each inoculated animal was introduced as the DR into a transmission cage with an immunologically naïve RC. Nasal washes were then collected every other day from each animal. (A to D) Viral shedding curves for IM mock-, HA-, NA-, or HA + NA–vaccinated DRs, respectively, and their paired RCs. DR shedding curves are shown in color, and RCs are shown with solid black lines. To determine whether vaccine-induced immunity altered viral shedding kinetics, analyses of viral titers were performed. (E) and (F) Peak nasal wash titers and total viral shedding by area under the curve (AUC) for IM vaccinated DRs, respectively. Orange, blue, pink, and green lines or symbols represent IM mock-, HA-, NA-, and HA + NA–vaccinated DRs, respectively. Nasal wash samples were titrated on Madin-Darby canine kidney cells (MDCK cells), and results are expressed as log₁₀ TCID₅₀/ml of nasal wash. Dashed horizontal line denotes limit of detection. ^*Significant differences were determined by Kruskal-Wallis test with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli post hoc (P < 0.05). For (E) and (F), horizontal line and error bars represent mean ± SEM, respectively, and unless significant differences are indicated, all other comparisons are not significant.

Fig. 3.. Airborne transmission of the 2009 H1N1 virus from IN vaccinated preimmune DRs to immunologically naïve RC ferrets.

IN mock-, HA-, NA-, and HA + NA–vaccinated ferrets (n = 4 per group) were IN inoculated with 1 × 10⁴ TCID₅₀ of the 2009 H1N1 virus. Twenty-four hours later, each inoculated animal was introduced as the DR into a transmission cage with an immunologically naïve RC. Nasal washes were then collected every other day from each animal. (A to D) Viral shedding curves for IN mock-, HA-, NA-, or HA + NA–vaccinated DRs, respectively, and their paired RCs. DR shedding curves are shown in color, and RCs are shown with solid black lines. In (D), two DRs had superimposed shedding curves. These curves were interweaved to assist with visualizing the data. (E) and (F) Peak nasal wash titers and total viral shedding by AUC for IN vaccinated DRs, respectively. Orange, dark blue, pink, and green represent IN mock-, HA-, NA-, and HA + NA–vaccinated DRs, respectively. Nasal wash samples were titrated on MDCK cells, and results are expressed as log₁₀ TCID₅₀/ml of nasal wash. Dashed horizontal line denotes limit of detection (1 log₁₀ TCID₅₀/ml). *Significant differences were determined by Kruskal-Wallis test with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli post hoc comparison (P < 0.05). For (E) and (F), horizontal line and error bars represent mean ± SEM, respectively, and unless significant differences are indicated, all other comparisons are not significant.

Fig. 4.. Evaluation of antibody responses after primary infection with reassortant viruses to selectively induce immunity to HA, NA, or HA and NA.

To induce immunity via infection, ferrets (n = 4 per group) were IN infected with 1 × 10⁶ TCID₅₀ of reassortant or wild-type recombinant viruses. (A) Schematic showing the gene segment composition of reassortant viruses used to induce immunity to H1N1 HA and/or NA on the 1968 H3N2 virus backbone [created in BioRender; T.C.S. (2025); https://BioRender.com/hht98j9]. On day 82 post–primary infection with reassortant viruses, serum was collected from the ferrets, and the antibody response was evaluated. (B) displays binding IgG antibody titers against H1 HA and N1 NA, while (C) shows HAI titers, neutralization titers, and NA activity inhibiting titers. Binding antibody titers were determined by ELISA. HAI and neutralization titers were determined via HAI assay and microneutralization assays, respectively. NA inhibition titers were determined via ELLA. ELISA, HAI, and ELLA assays were performed in duplicate, and microneutralization assays were performed with four technical replicates. For all antibody analyses, individual values are displayed with horizontal line, and error bars showing mean ± SEM, respectively. Antibody responses in ferrets infected with rsH1N2, rsH3N1, and rsH1N1 are shown in dark blue, pink, and green, respectively. Antibody responses in ferrets infected with wild-type 1968 H3N2 and 2009 H1N1 viruses are shown in orange and light blue, respectively. Horizontal dotted black lines denote limit of detection. ^*Different from 1968 H3N2 and rsH3N1 DRs (P < 0.02). ^†Different from 1968 H3N2 and rsH1N2 DRs (P < 0.042). ^#Different from 1968 H3N2 and rsH3N1 DRs (P < 0.05), determined via Kruskal-Wallis test with post hoc two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. For (B) and (C), unless significant differences are shown, all other comparisons are not significant.

Fig. 5.. Airborne transmission of the 2009 H1N1 virus from infection-induced preimmune DRs to immunologically naïve RC ferrets.

1968 H3N2–, rsH1N2-, rsH3N1-, rsH1N1-, and 2009 H1N1–preimmune ferrets were challenged with 1 × 10⁴ TCID₅₀ of the 2009 H1N1 virus and used as DRs to immunologically naïve RCs in airborne transmission studies. (A to E) Viral shedding curves for 1968 H3N2–, rsH1N2-, rsH3N1-, rsH1N1-, and 2009 H1N1–preimmune DRs, respectively, and their paired RCs. Viral titers from DR animals are shown in solid-colored lines, and RC shedding curves are shown in black. 1968 H3N2-, rsH1N2-, rsH3N1-, rsH1N1-, and 2009 H1N1-preimmune DRs are displayed in orange, dark blue, pink, green, and light blue, respectively. Results are expressed as log₁₀ TCID₅₀/ml of nasal wash. Dashed horizontal line denotes limit of detection. Analyses of viral titers were performed on (F) peak nasal wash titers and (G) total viral shedding (AUC). ^*Significantly different from indicated group (P < 0.011). Significant differences were determined by Kruskal-Wallis test with post hoc two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli for antibody analysis (P < 0.05). For (F) and (G), horizontal line and error bars represent mean ± SEM, respectively, and unless significant differences are indicated, all other comparisons are not significant. Two RC ferrets paired with 1968 H3N2 DRs developed a secondary infection after becoming infected and were removed from the study. Thus, data from one RC are displayed until day 6, while data from the second RC are displayed until day 10.

Fig. 6.. Substitutions HA E171K and HA G172E identified during transmission of the 2009 H1N1 virus from IM vaccinated preimmune DRs to immunologically naïve RC ferrets.

The relative frequency (y axis) of (A) HA E171K and (B) HA G172E present at ≥5% in at least one ferret nasal wash sample collected from transmission experiments with IM vaccinated ferrets. For all plots, data are grouped by vaccination status of the DR (across the top). The x axis represents days postinfection of the DR ferret. Variant frequencies for individual DR ferrets are shown with solid symbols with solid connecting lines, while individual RC ferrets are shown in open symbols with dashed connecting lines. Frequencies for mock-, HA-, NA-, and HA + NA–vaccinated DRs with their paired RC are shown in orange, dark blue, pink, and green, respectively. Dashed line indicates 0.05 frequency cutoff for sequence analysis. Solid line at 0.5 indicates 50% frequency above which nonsynonymous mutations are considered substitutions. Black circle and “S” on y axis denote frequency of the minor variant in the virus stock.

Fig. 7.. Substitutions HA E171K and HA G172E identified during transmission of the 2009 H1N1 virus from IN vaccinated preimmune DRs to immunologically naïve RC ferrets.

The relative frequency (y axis) of (A) HA E171K and (B) HA G172E present at ≥5% in at least one ferret nasal wash sample collected from transmission experiments with IN vaccinated ferrets. For all plots, data are grouped by vaccination status of the DR (across the top). The x axis represents days postinfection of the DR ferret. Variant frequencies for individual DR ferrets are shown in solid symbols with solid connecting lines, while individual RC ferrets are shown in open symbols with dashed connecting lines. Frequencies for mock-, HA-, NA-, and HA + NA–vaccinated DRs with their paired RC are shown in orange, dark blue, pink, and green, respectively. Dashed line indicates 0.05 frequency cutoff for sequence analysis. Solid line at 0.5 indicates 50% frequency above which nonsynonymous mutations are considered substitutions. Black circle and S on y axis denote frequency of the minor variant in the virus stock.

Fig. 8.. Substitutions HA E171K and HA G172E identified during transmission of the 2009 H1N1 virus from infection-induced preimmune DRs to immunologically naïve RC ferrets.

The relative frequency (y axis) of (A) HA E171K and (B) HA G172E present at ≥5% in at least one ferret nasal wash sample collected from transmission experiments with infection-induced preimmune DR ferrets. For all plots, data are grouped by immune status of the DR (across the top). The x axis represents days postinfection of the DR ferret. Variant frequencies for individual DR ferrets shown in solid symbols with solid connecting lines, while individual RC ferrets are shown in open symbols with dashed connecting lines. Frequencies for 1968 H3N2, rsH1N2, rsH3N1, and rsH1N1 preimmune DRs with their paired RCs are shown in orange, dark blue, pink, and green, respectively. No frequencies for H1N1-preimmune DRs and their RCs are reported, as infectious virus was not recovered from these animals. Dashed line indicates 0.05 frequency cutoff for sequence analysis. Solid line at 0.5 indicates 50% frequency above which nonsynonymous mutations are considered substitutions. Black circle and S on y axis denote frequency of the minor variant in the virus stock.

Fig. 9.. Analysis of the relationship between reductions in viral titer in DR ferrets and airborne transmission to RCs.

Using DR viral titer data and the TE across all experiments, we performed an aggregate analysis to assess the relationship between immune status, viral titer, and transmission. All viral titer data from DR ferrets were grouped categorically into immunity via infection, immunity via vaccination, or no immunity. Viral titers between these groups were then compared via one-way analysis of variance (ANOVA) with Tukey’s post hoc test. Subsequently, logistical regression was performed, and receiving operator characteristic (ROC) curves were generated to define the relationship between reduction in viral load and transmission. (A) Viral titers plotted by immune status on each day postinfection. (B) Logistical regression curves defining the relationship between reductions in viral load on days 1 and 3, and probability of transmission to contact animals. ^*Significantly different from immunity via infection (P < 0.001). ^†All groups significantly different from each other (P < 0.0001).