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Journal of Virology logoLink to Journal of Virology
. 2022 May 31;96(12):e00320-22. doi: 10.1128/jvi.00320-22

Reduction of Influenza A Virus Transmission in Mice by a Universal Intranasal Vaccine Candidate is Long-Lasting and Does Not Require Antibodies

Graeme E Price a,✉,#, Chia-Yun Lo a,#, Julia A Misplon a, Suzanne L Epstein a
Editor: Stacey Schultz-Cherryb
PMCID: PMC9215256  PMID: 35638848

ABSTRACT

Vaccination against influenza virus infection can protect the vaccinee and also reduce transmission to contacts. Not all types of vaccines induce sterilizing immunity via neutralizing antibodies; some instead permit low-level, transient infection. There has been concern that infection-permissive influenza vaccines may allow continued spread in the community despite minimizing symptoms in the vaccinee. We have explored that issue for a universal influenza vaccine candidate that protects recipients by inducing T cell responses and nonneutralizing antibodies. Using a mouse model, we have shown previously that an adenoviral vectored vaccine expressing nucleoprotein (NP) and matrix 2 (M2) provides broad protection against diverse strains and subtypes of influenza A viruses and reduces transmission to contacts in an antigen-specific manner. Here, we use this mouse model to further explore the mechanism and features of that reduction in transmission. Passive immunization did not reduce transmission from infected donors to naive contact animals to whom passive serum had been transferred. Vaccination of antibody-deficient mIgTg-JHD−/− mice, which have intact T cell responses and antigen presentation, reduced transmission in an antigen-specific manner, despite the presence of some virus in the lungs and nasal wash, pointing to a role for cellular immunity. Vaccination at ages ranging from 8 to 60 weeks was able to achieve reduction in transmission. Finally, the immune-mediated reduction in transmission persisted for at least a year after a single-dose intranasal vaccination. Thus, this infection-permissive vaccine reduces virus transmission in a long-lasting manner that does not require antibodies.

IMPORTANCE Universal influenza virus vaccines targeting antigens conserved among influenza A virus strains can protect from severe disease but do not necessarily prevent infection. Despite allowing low-level infection, intranasal immunization with adenovirus vectors expressing the conserved antigens influenza nucleoprotein (A/NP) and M2 reduces influenza virus transmission from vaccinated to unvaccinated contact mice. Here, we show that antibodies are not required for this transmission reduction, suggesting a role for T cells. We also show that transmission blocking could be achieved in recipients of different ages and remained effective for at least a year following a single-dose vaccination. Such vaccines could have major public health impacts by limiting viral transmission in the community.

KEYWORDS: influenza, T cells, transmission, universal vaccine, antibody, intranasal, mouse model

INTRODUCTION

Novel influenza viruses remain a substantial public health concern, with new viruses occasionally emerging and becoming readily transmissible in humans. Conventional influenza vaccines induce antibodies to the antigenically variable hemagglutinin (HA) and neuraminidase (NA) glycoproteins. Responses include neutralizing antibodies to HA that block viral entry, preventing infection and, if this blocking is complete, preventing transmission to others. Reduction of influenza virus transmission by prior infection or conventional vaccines has been studied both in animal models and in humans. It was shown in the 1960s that previous infection with live influenza virus not only protected mice against viral challenge but reduced transmission to contacts (1). In guinea pigs, live attenuated vaccine was more effective than inactivated vaccine in reducing transmission (2). A study in ferrets showed that live attenuated vaccine reduced transmission of virus matched to the vaccine but not mismatched virus (3). In chickens, H7 vaccines reduced transmission (4). In humans, an epidemiological study of children attending daycare found that vaccination reduced influenza morbidity in their household contacts (5). Similarly, a study suggested that vaccination of schoolchildren reduced transmission to staff and unvaccinated children (6).

However, conventional HA-based vaccines have significant disadvantages. They must be matched to circulating strains to be optimally effective (7), and manufacture takes several months after a strain has been identified (8). Consequently, strain-matched vaccines would not be available for a long time after a novel strain emerges. In contrast, immune responses directed against conserved antigens can provide cross-protection against different influenza viruses regardless of their HA and NA strain or even across subtypes (9–11). Thus, these antigens offer new vaccine targets, and vaccines based on them might be used off-the-shelf at the time of an unexpected outbreak or at other times to supplement strain-matched vaccines. Conserved target antigens include nucleoprotein (NP), matrix, and the stem region of HA.

While the HA stem is much more conserved than the immunodominant globular HA head, the stem region differs between group 1 and group 2 viruses, and group-specific imprinting (12) might reduce responsiveness to a stem-based vaccine. NP and matrix 2 (M2) are highly conserved between influenza A viruses even from different subtypes and groups (13).

We have previously demonstrated that a universal vaccine candidate based on recombinant adenovirus (rAd) vectors expressing NP and M2 (A/NP+M2-rAd) protects vaccinated mice and ferrets against lethal challenge with various influenza virus strains, including highly pathogenic H5N1 (11, 14). Protection against morbidity and mortality is induced rapidly, as well as a reduction in virus titers in the respiratory tract, compared to animals vaccinated with control vectors. Mechanism studies showed that while the A/NP+M2-rAd vaccine does not induce neutralizing antibodies (15), both T cells and nonneutralizing antibodies play roles in protection (16, 17).

However, a perceived weakness of such a conserved antigen vaccine is that because it does not induce neutralizing antibodies, but rather permits limited infection, vaccinated individuals who become infected might still transmit virus to others (18). In a series of studies, we have addressed the effect of this type of immunity on influenza A virus transmission in a mouse model (15, 19). We have shown that single-dose, intranasal A/NP+M2-rAd vaccination significantly reduces viral transmission from vaccinated and subsequently infected mice to naive contacts. Reduction in transmission is antigen specific and is more effective for intranasal than intramuscular vaccination. Vaccination reduces transmission by both direct contact and airborne routes.

In this report, we have extended those studies to examine the role of antibodies and T cells in transmission reduction, to study transmission by vaccinated and subsequently infected donors of different ages, and to test the durability of transmission reduction by this vaccination.

RESULTS

The effect of passive serum transfer in reduction of influenza transmission.

We know from previous studies that the reduction of influenza A virus transmission by prior rAd immunization of donors is due to immunologically specific responses, not innate immunity to the Ad vector, since transmission is reduced by prior immunization with A/NP+M2-rAd but not by B/NP-rAd. Thus, the reduction in transmission presumably depends on antigen-specific antibodies, T cells, or a combination of both. As an initial step to assess which immune effector mechanisms are involved, we tested the ability of passive antibodies to reduce transmission in the absence of an antigen-specific T cell response. Serum was collected from BALB/c mice immunized intranasally (i.n.) with A/NP+M2-, A/NP-, M2-, or B/NP-rAd and from unimmunized (naive) mice every 2 weeks from about 4 to 32 weeks after immunization. These sera were then pooled for each immunization group. The resulting serum pools were tested by enzyme-linked immunosorbent assay (ELISA) for IgG antibodies reactive with influenza A/Puerto Rico/8/34 nucleoprotein (A/NP), M2 ectodomain (M2e), and influenza B/Ann Arbor/1/86 virus nucleoprotein (B/NP). Results showed high titers of IgG antibodies to the respective antigens but little or no activity on mismatched antigens (Fig. 1A). When tested for IgA antibodies, only the B/NP serum showed activity above background (Fig. 1B).

FIG 1.

FIG 1

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Antibody levels in serum pools and in serum of mice after passive transfer. Serum pools were obtained by sequentially bleeding unimmunized (naive) BALB/c mice or BALB/c immunized i.n. with 5 × 109 particles each of A/NP-rAd and M2-rAd, 5 × 109 particles of A/NP-rAd alone or M2-rAd alone, or 1 × 1010 particles of B/NP-rAd. The pooled sera before the transfer (input serum pools) were tested for IgG (A) and IgA (B) by ELISA on plates coated with A/NP, M2e, or B/NP as indicated. Serum and BAL specimens from recipient BALB/c mice (n = 5 per group) were collected 24 h after passive immunization (serum transfer), and samples were pooled prior to testing for IgG (C) and IgA (D) by ELISA against A/NP, M2e, and B/NP as indicated. Control sera and BAL specimens collected from separate BALB/c mice 4 weeks postvaccination with A/NP+M2- or B/NP-rAd were used for comparison. Bars show area under the curve for absorbance versus dilution for each sample.

To test transmission reduction, naive BALB/c mice were injected intraperitoneally (i.p.) with 1 mL of a serum pool 1 day prior to A/Udorn/307/72 (H3N2) (A/Udorn) challenge. These infected passively immunized mice were then placed in contact with naive Carworth Farms White (CFW) contact animals 24 h after A/Udorn challenge. To assess antibody titers achieved in vivo, representative recipient mice were bled and bronchoalveolar lavage (BAL) specimens were collected 24 h after serum transfer. When tested by ELISA, these sera showed the same specificity of IgG antibodies as the input serum pools but with approximately a 2- to 3-fold decrease in antibody level (compare Fig. 1A and C). No IgG antibodies were seen in BAL specimens (Fig. 1C), and no IgA was detected in either serum or BAL specimens of recipients (Fig. 1D). IgG levels in serum remained similar on days 0 to 4 following transfer (Fig. 2).

FIG 2.

FIG 2

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Serum IgG levels remain constant for 5 days post passive serum transfer. Naive BALB/c mice were injected i.p. with 1 mL of the indicated input serum pools on day −1. Sera were collected from recipient mice (n = 5 per group) starting 24 h posttransfer (day 0) and pooled prior to testing for IgG by ELISA against A/NP, M2e, and B/NP. As a control, serum was collected on the same days as the serum recipients from separate BALB/c mice vaccinated with A/NP+M2- or B/NP-rAd 4 weeks earlier. Note that mice were infected with 104 TCID50 of A/Udorn immediately after collection of the day 0 sample. Data points show area under the curve for absorbance versus dilution for each sample.

Mice actively immunized i.n. with A/NP+M2-rAd or B/NP-rAd were used as controls in the transmission experiments. As shown in Fig. 1C (left) vaccinated controls had high levels of antigen-specific IgG in both serum samples and BAL specimens, as expected for responses to intranasal administration of the vaccine. They also had considerable IgA responses in their BAL specimens but not in serum (Fig. 1D).

The kinetics of virus titers in lung and nasal wash samples were assessed (Fig. 3). Active immunization with A/NP+M2-Ad reduced lung titers to undetectable on all days and nasal wash titers to close to the limit of detection. Overall, passive transfer of serum had little effect. Viral titers in mice receiving serum specific for A/NP or B/NP were similar to those for naive serum. Anti-A/NP+M2-rAd or anti-M2-rAd serum significantly reduced lung virus titers relative to naive serum but far less than active immunization did. None of the passive immunizations had a clear effect on nasal wash titers.

FIG 3.

FIG 3

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Virus kinetics after passive serum transfer. Mice were infected with 104 TCID50 of A/Udorn 24 h after serum transfer (or 4 weeks post-rAd vaccination). Virus titers were assessed in lungs (left) and nasal washes (right) collected from groups of mice treated as indicated in the legend. Data show mean ± standard error of the mean (SEM) from groups of 5 mice on days 1 to 3 or 12 mice on day 4. Nasal wash titers were not assessed on day 3 for A/NP+M2-rAd- or B/NP-rAd-vaccinated controls. The dashed horizontal black line indicates assay limit of detection. Differences at each time point were assessed by one way ANOVA against naive serum as a control group for the corresponding site (lung or nasal wash) and the corresponding day postchallenge; *, significant difference (P < 0.05).

Despite the reduction in lung viral titers in some groups, there was no significant reduction in transmission from passively immunized donor mice. In contrast, there was 90% reduction in transmission from control mice actively immunized with A/NP+M2-rAd (Table 1). The results do not rule out a role of antibodies; several possible explanations will be mentioned in Discussion.

TABLE 1.

Effect of passive serum transfer on virus transmission

Donor treatment No. of infected BALB/c donors at day 4/total no. of donors (mean titer ± SEM)
 No. of infected CFW contacts/total no. of contacts (mean titer ± SEM)
 % Reduction in transmission vs B/NP-rAda
Lung (log10TCID50/mL) Nasal wash (Log10TCID50/mL) Total (%) Lung (log10TCID50/mL) Nasal wash (Log10TCID50/mL) Total (%)
A/NP+M2 serum 11/12 (4.57 ± 0.38) 9/12 (2.95 ± 0.12) 12/12 (100) 4/18 (4.41 ± 1.18) 8/18 (3.16 ± 0.29) 8/18 (44.4) 20 (NS)
A/NP serum 12/12 (6.32 ± 0.11) 12/12 (3.28 ± 0.16) 12/12 (100) 8/18 (5.90 ± 0.36) 8/18 (3.50 ± 0.27) 9/18 (50.0) 10 (NS)
M2 serum 11/12 (4.68 ± 0.50) 11/12 (3.59 ± 0.28) 12/12 (100) 8/18 (4.39 ± 0.53) 10/18 (3.92 ± 0.22) 11/18 (61.1) 0 (NS)
B/NP serum 12/12 (6.38 ± 0.10) 11/12 (3.56 ± 0.19) 12/12 (100) 7/18 (5.09 ± 0.63) 7/18 (4.05 ± 0.46) 9/18 (50.0) 10 (NS)
Naïve serum 12/12 (6.43 ± 0.12) 12/12 (3.32 ± 0.18) 12/12 (100) 7/18 (4.95 ± 0.60) 7/18 (3.48 ± 0.38) 8/18 (44.4) 20 (NS)
A/NP+M2-rAd i.n. 0/12 0/12 0/12 (0) 1/18 (5.03) 0/18 1/18 (5.6) 90 (P < 0.005)
B/NP-rAd i.n. 11/11 (6.73 ± 0.12) 11/11 (4.19 ± 0.25) 11/11 (100) 3/18 (5.06 ± 0.56) 10/18 (4.01 ± 0.36) 10/18 (55.5)
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a

NS, not significant.

The effect of cellular immunity in reduction of influenza transmission.

We investigated whether virus-specific cellular immune responses are sufficient to mediate the reduction in transmission from A/NP+M2-rAd-immunized mice. In initial experiments, we depleted T cells from A/NP+M2-rAd- and B/NP-rAd-immunized mice, infected them with influenza virus, and then used these mice as donors in transmission experiments. While >90% depletion of CD4+, CD8+, and CD90+ cells from both lung and spleen was achieved, transmission results were inconclusive.

As an indirect approach to assess the role of T cells in transmission reduction, we compared transmission from wild-type, fully immunocompetent BALB/c donors to transmission from mIgTg-JHD−/− donors that are T cell competent but antibody deficient. BALB/c and mIgTg-JHD−/− mice were intranasally immunized with A/NP+M2-rAd or B/NP-rAd. Peptide-specific lung T cell responses were assessed by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay 1 month later. Responses to the immunodominant H2-Kd-restricted NP147–155 epitope were seen in the lungs of A/NP+M2-rAd-immunized BALB/c and mIgTg-JHD−/− mice (Fig. 4A and B). Responses against subdominant T cell epitopes NP55–69 and M2e2–24 in BALB/c mice have been observed in previous studies (11, 20, 21), but in this study, they were variable and low relative to NP147–155.

FIG 4.

FIG 4

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Antibody and T cell responses to vaccination in mIgTg-JHD−/− mice versus BALB/c mice. mIgTg-JHD−/− and control BALB/c mice were immunized i.n. with 5 × 109 particles each of A/NP-rAd and M2-rAd or 1 × 1010 particles of B/NP-rAd. Immune responses were assessed 4 weeks postimmunization. (A, B) T cell responses in the lung were assessed by IFN-γ ELISPOT following stimulation with the indicated peptides. Data show mean ± SEM for 3 mice, expressed in terms of frequency (A) and total cells per organ (B). Differences for each mouse strain-vaccine combination were assessed by one way ANOVA against the appropriate no peptide control; *, significantly different from no peptide control (P < 0.05). (C) Serum IgG antibody responses against A/NP, M2e, and B/NP are shown for pooled sera; bars show area under the curve for absorbance versus dilution for each sample and confirm the lack of antibody responses in mIgTg-JHD−/− mice.

BALB/c mice made strong serum IgG responses against A/NP and M2e or B/NP when immunized with A/NP+M2-rAd or B/NP-rAd, respectively, but no serum IgG responses were detectable in immunized mIgTg-JHD−/− mice (Fig. 4C). Thus, as expected, mIgTg-JHD−/− mice make antigen-specific T cell responses but not antibodies following A/NP+M2-rAd immunization.

One month after immunization, A/NP+M2-rAd- or B/NP-rAd-immunized BALB/c and mIgTg-JHD−/− donors were infected with 104 50% tissue culture infective dose (TCID50) of A/Udorn and the next day placed in contact with naive CFW mice. As before, transmission was detected by virus titration of lung homogenates and nasal washes from contact mice on day 3 after contact. Lung and nasal wash virus titers were also assessed in parallel groups of donors on days 1, 2, 3, and 4 after infection to monitor clearance kinetics (Fig. 5).

FIG 5.

FIG 5

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Virus kinetics in vaccinated mIgTg-JHD−/− mice and BALB/c mice. mIgTg-JHD−/− and control BALB/c mice were immunized i.n. with 5 × 109 particles each of A/NP-rAd and M2-rAd or 1 × 1010 particles of B/NP-rAd. Four weeks postimmunization, mice were infected with 104 TCID50 of A/Udorn, and virus titers were assessed in lungs (left) and nasal washes (right) at the indicated time points. Virus titration data show mean ± SEM from groups of 3 at days 1 to 3 and 22 on day 4 for mIgTg-JHD−/− mice and for groups of 5 at days 1 to 3 and 16 on day 4 for BALB/c mice. The dashed horizontal black line shows assay limit of detection. Differences between A/NP+M2-rAd and B/NP-rAd immunization were assessed for each mouse strain by t test. Differences are denoted as follows: BALB/c *, P < 0.001; #, P < 0.005; ‡, P < 0.02; mIgTg-JHD−/− §, P < 0.001; †, P < 0.005; ¶, P < 0.05.

Lung virus titers increased in B/NP-rAd-immunized donors of both mouse strains until day 3. Lung titers were lower in the groups receiving A/NP+M2-rAd vaccine than in B/NP-rAd controls, significantly so on all days for BALB/c and on days 3 and 4 for mIgTg-JHD−/− mice. Of the A/NP+M2-rAd-immunized groups, BALB/c mice had lung virus titers near baseline, lower on all days than those of mIgTg-JHD−/− mice. Nasal wash titers were also reduced by A/NP+M2-rAd immunization, and again titers were lower in BALB/c. Overall, this data shows that mIgTg-JHD−/− mice, able to mount T cell but not B cell responses, can partially control virus replication in the respiratory tract following i.n. A/NP+M2-rAd immunization. This inhibition of viral replication is antigen specific, as it was not observed following B/NP-rAd immunization. However, vaccination reduced virus replication more rapidly and to a greater extent in fully immunocompetent BALB/c mice.

When virus transmission was examined (Table 2), as previously seen (Table 1), transmission from A/NP+M2-rAd-immunized BALB/c donors to unimmunized CFW contacts was significantly reduced (by 95.2% [P < 0.001]) compared with B/NP-rAd-immunized donors. For unknown reasons, there was less transmission from B/NP-rAd-immunized mIgTg-JHD−/− than from B/NP-rAd-immunized BALB/c donors (54.6% versus 87.5%, respectively). However, vaccination with A/NP+M2-rAd eliminated transmission from mIgTg-JHD−/− donors completely (Table 2), despite the fact that A/NP+M2-rAd-immunized mIgTg-JHD−/− donors had more virus in lungs or nasal washes than A/NP+M2-rAd-immunized BALB/c donors (Fig. 5). The data clearly indicate that, in the presence of strong antigen-specific T cell responses, antibodies are not required for the prevention of virus transmission from infected animals.

TABLE 2.

Transmission reduction from antibody-deficient immunized mice

Mouse strain Donor immunization No. of infected BALB/c donors at day 4/total no. of donors (mean titer ± SEM)
 No. of infected CFW contacts/total no. of contacts (mean titer ± SEM)
 % Reduction in transmission vs B/NP-rAd
Lung (log10TCID50/mL) Nasal wash (Log10TCID50/mL) Total (%) Lung (log10TCID50/mL) Nasal wash (Log10TCID50/mL) Total (%)
BALB/c A/NP+M2-rAd 1/16 (2.45) 4/16 (2.51 ± 0.06) 5/16 (31.3) 1/24 (2.45) 0/24 1/24 (4.2) 95.2 (P < 0.001)
B/NP-rAd 16/16 (6.27 ± 0.11) 16/16 (3.77 ± 0.17) 16/16 (100) 9/24 (4.95 ± 0.54) 21/24 (3.89 ± 0.24) 21/24 (87.5)
mIgTg-JHD−/− A/NP+M2-rAd 20/22 (4.44 ± 0.32) 20/22 (3.10 ± 0.12) 22/22 (100) 0/33 0/33 0/33 (0) 100 (P < 0.001)
B/NP-rAd 22/22 (6.00 ± 0.22) 22/22 (4.28 ± 0.17) 22/22 (100) 7/33 (4.15 ± 0.44) 15/33 (3.36 ± 0.19) 18/33 (54.6)
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Antibody and T cell responses after single dose mucosal vaccination at different ages and durability of transmission reduction.

BALB/c mice were vaccinated with A/NP+M2-rAd or B/NP-rAd intranasally at 8, 12, or 60 weeks of age. These immunizations were done at staggered dates to permit simultaneous harvesting and testing of samples and simultaneous analysis of transmission to minimize variability. The mice immunized at 8 weeks provide a control that matches the standard regimen used in our previous studies but cannot be directly compared to the group immunized at 12 weeks. At different intervals after immunization (4 weeks or 1 year), serum samples, BAL samples, and lung cells were collected. Antibody and T cell responses of 3 mice per group were assessed by ELISA and ELISPOT assay, with all samples tested simultaneously in each assay. As shown in Fig. 6A, all 3 groups had substantial serum IgG antibody levels specific for the immunizing antigens. IgG antibodies to all 3 vaccine antigens were also present in BAL specimens at somewhat lower titers (Fig. 6C). IgG responses to M2 were weaker in both serum and BAL specimens than to A/NP or B/NP. IgA antibodies were marginal in serum but detectable in BAL specimens (Fig. 6B and D). In the group immunized at 12 weeks and tested at 64 weeks, IgA antibodies to A/NP were lower than in mice immunized 4 weeks earlier, which might suggest waning of the IgA response over the long interval (1 year) between immunization and BAL specimen collection.

FIG 6.

FIG 6

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Antibody responses following vaccination at different ages and intervals. BALB/c mice were immunized i.n. with 5 × 109 particles each of A/NP-rAd and M2-rAd or 1 × 1010 particles of B/NP-rAd at different ages as indicated. At 52 or 4 weeks after immunization, IgG responses in serum (A) and BAL specimens (C) and IgA responses in serum (B) and BAL specimens (D) against A/NP, M2e, and B/NP were assessed by ELISA. Bars show mean area under the curve for absorbance versus dilution for each sample ± SEM from groups of 3 mice. Differences between A/NP+M2-rAd and B/NP-rAd immunization were assessed by t test for mice with the same immunization age and challenge interval, with significant differences denoted as follows: *, P < 0.001; #, P < 0.005; †, P < 0.01; ‡, P < 0.05.

T cell responses assessed by IFN-γ ELISPOT testing of lung cells are shown in Fig. 7A and B. All 3 A/NP+M2-rAd groups responded strongly to the dominant CD8 epitope NP147–155 with lower responses against NP55–69 and M2e2–24. In this simultaneous testing, the frequency and total number of responding cells per organ were somewhat higher for the groups tested at 64 weeks of age, whether that was 4 weeks or 1 year postimmunization. This indicates that lung T cell responses persist for at least 1 year postimmunization, and that the ability to mount strong responses to rAd vaccination is not restricted to young animals.

FIG 7.

FIG 7

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T cell responses following vaccination at different ages and intervals. BALB/c mice were immunized i.n. with 5 × 109 particles each of A/NP-rAd and M2-rAd or 1 × 1010 particles of B/NP-rAd at different ages, as indicated. At 52 or 4 weeks after immunization, T cell responses in lungs of all groups were assessed by IFN-γ ELISPOT following restimulation with the indicated peptides in a single experiment. Results shown are the mean ± SEM from groups of 3 mice expressed in terms of frequency (A) and total cells per organ (B). Differences for each mouse age-vaccine combination were assessed by one way ANOVA against the appropriate no peptide control; *, significantly different from no peptide control (P < 0.05).

Additional mice in each group were infected with A/Udorn and used to assess viral kinetics (Fig. 8) or as donors in transmission experiments (Table 3). Strikingly, no virus was detected in lung homogenates of A/NP+M2-rAd-immunized mice in any of the groups, indicating effective control of A/Udorn infection in the lower respiratory tract even a year after immunization (P < 0.01 relative to age-matched B/NP-rAd controls). Nasal wash titers were also effectively controlled in young mice immunized with A/NP+M2-rAd (P < 0.02). Nasal wash titers in aged A/NP+M2-rAd-immunized mice were higher at day 2 but effectively controlled by day 4, while viral titers remained high in matched B/NP-rAd-immunized animals.

FIG 8.

FIG 8

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Virus titer following vaccination at different ages and intervals. BALB/c mice were immunized i.n. with 5 × 109 particles each of A/NP-rAd and M2-rAd or 1 × 1010 particles of B/NP-rAd at different ages as indicated. Challenge with 104 TCID50 of A/Udorn was conducted simultaneously for all groups 52 or 4 weeks after immunization, and virus titers were assessed in lungs (left) and nasal washes (right) at the indicated time points. Data show mean ± SEM from groups of 3 to 5 mice at days 1 to 3 and 12 mice at day 4. For groups immunized at 60 weeks of age, enough mice were available for testing only on days 2 and 4. Dashed line shows assay limit of detection. Differences between A/NP+M2-rAd and B/NP-rAd immunization were assessed by t test for mice with the same immunization age and challenge interval. Differences are denoted as follows: *, P < 0.001; #, P < 0.005; †, P < 0.01; ‡, P < 0.02.

TABLE 3.

Durability of transmission reduction in mice immunized at different ages

Donor treatment (ages immunized, challenged) No. of infected BALB/c donors at day 4/total no. of donors (mean titer ± SEM)
 No. of infected CFW contacts/total no. of contacts (mean titer ± SEM)
 % Reduction in transmission vs B/NP-rAd
Lung (log10TCID50/mL) Nasal wash (Log10TCID50/mL) Total (%) Lung (log10TCID50/mL) Nasal wash (Log10TCID50/mL) Total (%)
A/NP+M2-rAd i.n. (12 wk, 64 wk) 0/12 7/12 (2.47 ± 0.04) 7/12 (58.3) 0/18 1/18 (2.69) 1/18 (5.6) 92.9 (P < 0.001)
B/NP-rAd i.n. (12 wk, 64 wk) 12/12 (6.01 ± 0.18) 12/12 (4.36 ± 0.22) 12/12 (100) 9/18 (5.21 ± 0.46) 11/18 (3.60 ± 0.28) 14/18 (77.8)
A/NP+M2-rAd i.n. (60 wk, 64 wk)
 0/12 4/12 (2.88 ± 0.19) 4/12 (33.3) 2/18 (5.07 ± 0.38) 3/18 (3.53 ± 0.51) 3/18 (16.7) 76.9 (P < 0.005)
B/NP-rAd i.n. (60 wk, 64 wk) 12/12 (6.20 ± 0.17) 12/12 (4.49 ± 0.17) 12/12 (100) 7/18 (5.25 ± 0.37) 13/18 (3.88 ± 0.29) 13/18 (72.2)
A/NP+M2 rAd i.n. (8 wk, 12 wk) 0/12 3/12 (2.45 ± 0.00) 3/12 (25.0) 0/18 0/18 0/18 (0.0) 100 (P < 0.001)
B/NP-rAd i.n. (8 wk, 12 wk) 12/12 (6.13 ± 0.12) 12/12 (4.17 ± 0.20) 12/12 (100) 2/18 (5.61 ± 0.58) 11/18 (3.99 ± 0.38) 12/18 (66.7)
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As shown in Table 3, mice immunized with A/NP+M2-rAd when young (8 weeks old) and challenged 4 weeks later had reduced transmission (100% transmission reduction compared to that of age-matched B/NP controls). Mice immunized with A/NP+M2-rAd when 12 weeks old and challenged 1 year later had 92.9% reduction compared to that of B/NP-rAd controls. Mice immunized with A/NP+M2-rAd when 60 weeks old and challenged 4 weeks later had 76.9% reduction compared to that of B/NP controls, which is not as complete but still quite effective. Thus, reduction in transmission from vaccinated mice can last at least 1 year. In addition, immunization of mice ~1 year old is still effective in reducing transmission.

DISCUSSION

Universal influenza vaccines have the potential to protect against morbidity and mortality from a wider range of virus strains than vaccines inducing neutralizing antibodies to HA. However, vaccines acting via T cells and nonneutralizing antibodies may permit mild, transient infection (so called infection-permissive vaccines). For that reason, transmission of virus from vaccinated individuals who become infected has been raised as a concern (18). Studies in animal models can address this concern about transmission to others and allow analyses that would be difficult in humans or large animal models, including identification of immune mechanisms responsible for control of transmission. We have previously shown that for mice given universal vaccine A/NP+M2-rAd i.n. and challenged 1 month later, transmission to contacts was greatly reduced (15, 19). In this study, we use the mouse model to examine the roles of antibodies and T cells in transmission reduction and to test durability of transmission reduction.

Passive transfer of serum from A/NP+M2-rAd- or M2-rAd-immunized mice resulted in significant reduction of lung viral titers, indicating that sufficient M2-specific IgG was present to exert an antiviral effect. However, serum from A/NP-rAd-immunized mice had no apparent effect on virus titer. While others (22, 23) have reported protection mediated by passive transfer of anti-A/NP IgG, differences in form of immunizing antigen, mouse strain, challenge virus, and assay time points make direct comparisons difficult. It is possible that the different outcome observed here could be explained by quantitative and/or qualitative (e.g., antibody isotype or affinity) differences in the anti-A/NP sera.

Passively transferred serum antibody was inadequate to reduce transmission. This result does not rule out participation of antibodies in reducing transmission. First, antibodies alone may not be sufficient without participation of additional mechanisms. Second, serum titers achieved by passive transfer were 4-fold lower than in actively immunized animals. Third, local antibodies including IgA are induced in the respiratory tract by active immunization but are lacking in recipients of passive antibody. Antibody isotype and localization may be important for effective virus clearance and blocking of transmission. Passive transfer of IgA against HA has been shown to prevent transmission in guinea pigs (24), but in our experiments, IgA against M2 or A/NP was not present in the transferred serum.

To test the possible role of T cells in transmission reduction and independence of local antibody responses, we vaccinated transgenic mIgTg-JHD−/− mice. These mice are unable to produce any antigen-specific antibodies due to a disrupted heavy chain J segment locus. They have an inserted transgene for a membrane IgM, permitting B cell maturation and thus antigen presentation by B cells in support of normal T cell responses. While transmission rates were lower from B/NP-rAd-immunized mIgTg-JHD−/− donors than from BALB/c donors, transmission was still substantial and was abolished in an antigen-specific manner by vaccination. Thus, cellular immunity can suffice in this scenario and antibody is not required for reduction in transmission by vaccination.

Interestingly, although mIgTg-JHD−/− mice transmitted infection less effectively, they had higher levels of replicating virus in their lungs and nasal washes than vaccinated and infected BALB/c mice. There is precedent in other studies for lack of correlation between viral load in the nose or lungs and transmission (25, 26), and a variety of explanations have been suggested. For example, sequestered virus, which is not available for transmission, might be released from foci of infection during tissue homogenization.

Many experiments in the vaccination field are performed in young adult mice, which may not provide an adequate model of the various ages in the human population. In an effort to address this, we studied effects on transmission from animals vaccinated at 60 weeks of age. In both 8-week-old and 60-week-old mice, immune responses to the intranasal vaccination were strong and antigen specific. When those mice were challenged and used as donors, transmission reduction was somewhat less effective in the mice vaccinated at 60 weeks of age than at 8 weeks, but most transmission was still inhibited (76.9% reduction in 60 weeks versus 100% reduction in 8 weeks vaccinated mice).

Another limitation in many vaccine studies is that virus challenge is performed only a few weeks after immunization, thus not assessing the longevity of protection. In previous studies, we have shown that A/NP+M2-rAd vaccination provides durable protection, as shown by lethal challenge with diverse virus strains as long as a year after a single intranasal dose (13). Here, we add the finding that reduction in transmission is also durable. Mice given a single intranasal dose of vaccine at 12 weeks of age and challenged a year later still had a 92.9% reduction in transmission, relative to matched B/NP-rAd-immunized controls. The transmission rate in the long-term B/NP-rAd control group was just as high or higher as in the other groups tested for transmission after an interval of 1 month.

B/NP and the HA stem are additional target antigens that can be used to protect against diverse influenza virus strains (13, 27–29). Vaccine components providing A/NP and M2 could be combined with constructs providing B/NP and the HA stem so long as antigenic interference between the components is ruled out. Recombinant adenoviral vectors were used here, but other vaccine platforms could be used for expression of the target antigens.

There is great current interest in whether vaccines not only protect the vaccinated individual but also reduce disease transmission to others, a focus of attention during the ongoing COVID-19 pandemic. Reduction in severe disease and death of vaccinated individuals is the initial goal for a vaccine and can be established for humans directly by results of clinical trials. Reduction in transmission from vaccinated individuals experiencing breakthrough infections would provide additional benefits to the community but is much more difficult to demonstrate, requiring complicated follow-up of trial participants and their contacts and/or surveillance of large population cohorts. However, appropriate animal models can be valuable for providing proof of concept for transmission reduction and might inform aspects of design for human studies (30). Animal transmission studies may be particularly valuable for new vaccine technologies or antiviral drugs since little is known about their ability to control transmission.

For a universal influenza vaccine to achieve the goal of controlling unexpected outbreaks or pandemics, it will need several properties. As recommended by the National Institute of Allergy and Infectious Diseases (NIAID), it should protect broadly against group 1 and group 2 influenza strains and should protect for at least a year in different populations, such as different age groups (31). As they pointed out, an added benefit might be that such a vaccine may reduce transmission. The findings shown here provide proof of concept that a universal influenza vaccine candidate based on conserved antigens such as A/NP and M2, administered mucosally and not inducing neutralizing antibodies, can reduce influenza transmission in an animal model.

MATERIALS AND METHODS

Experimental animals and animal housing.

Female BALB/cAnNCR (BALB/c) and CFW mice [Crl:CFW(SW)] of 7 to 8 weeks old were purchased from Charles River Laboratories. Mice transgenic for a rearranged, irrelevant ((4-hydroxy-3-nitrophenyl)-acetyl specific) antibody VDJ segment fused to an IgM heavy chain lacking the secretory exon and poly-(A) sequence (mIgTg) (32), originally generated by Mark Shlomchik (Yale University, New Haven, CT), were maintained by heterozygous crossing onto the BALB/c-JHD−/− background at the Center for Biologics Research and Evaluation (CBER) animal facility. The resulting mIgTg-JHD−/− animals have an intact T cell compartment and have B cells that can act as antigen-presenting cells but cannot mount an antibody response. mIgTg-JHD−/− mice were immunized at 7 to 23 weeks of age in groups balanced between vaccine and control vectors to contain mice of the same age range. All animal experiments were conducted under animal biosafety level 2 conditions in animal facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), with all animal experiments and procedures approved by FDA White Oak Institutional Animal Care and Use Committee.

rAd vaccines.

Replication-deficient recombinant adenovirus (rAd) vectors expressing influenza A/Puerto Rico/8/34 nucleoprotein (A/NP), the consensus M2 sequence of human influenza A viruses (M2), or influenza B/Ann Arbor/1/86 virus nucleoprotein (B/NP) have been previously described (16, 17). Mice were immunized with either 5 × 109 particles each of A/NP-rAd and M2-rAd, 5 × 109 particles of A/NP-rAd alone or M2-rAd alone, or 1 × 1010 particles of B/NP-rAd given intranasally (i.n.) under isoflurane anesthesia in a 50-μL volume.

Nasal wash, BAL, blood, and lung sampling.

Mice were euthanized by overdose with ketamine (300 mg/kg of body weight)-xylazine (60 mg/kg). Following euthanasia, blood for serological assays was collected from the renal artery into microtainer SST tubes (Becton, Dickinson). Nasal wash collection (200 μL/mouse) and lung harvest were performed as described (15). Mucosal sampling for bronchoalveolar lavage (BAL) and lung cells was carried out as previously (14).

Influenza virus and virologic analyses.

Influenza virus A/Udorn/307/72 (H3N2) was propagated in eggs as previously described (33). Lungs were homogenized in 1 mL of L-15 medium as described previously (19). Virus titers in lung tissues and nasal wash samples were determined by TCID50 on MDCK cells and read out by hemagglutination with 0.5% chicken erythrocytes as previously described (11, 34), Titers were calculated by the method of moving averages and Weil’s tables (35, 36), with an assay limit of detection of 102.19 TCID50/mL.

Transmission experiments.

Transmission from vaccinated and subsequently infected mice (donors) to naive contact mice was assessed based on the model developed by Schulman and Kilbourne (37) and used in our previous studies (15, 19). Briefly, donors were infected with 104 TCID50 i.n. of A/Udorn in a 50-μL volume under isoflurane anesthesia. One day after infection, two infected donors were moved into each standard shoebox cage containing 3 naive contact animals. These cages were then placed on an open-sided, unventilated rack. On day 3 after cocaging (day 4 postinfection of donors), all mice were euthanized and lungs and nasal washes were collected. Contact animals were deemed positive for virus transmission if virus was detected by TCID50 assay in either lung homogenate, nasal wash, or both. Lung and nasal wash virus titers were also assessed in parallel groups of separately housed donors on days 1, 2, and 3 after infection to monitor clearance kinetics.

Passive antibody transfer experiments.

Blood was collected from rAd-immunized BALB/c mice via serial bleed from the tail or by submandibular veins at 2-week intervals. Serum was collected, pooled, and stored at −20°C without heat inactivation. Pooled sera before and after the transfer were tested for antigen specificity. One day prior to infection, naive BALB/c mice were injected intraperitonially (i.p.) with 1 mL of the appropriate serum pool.

Peptides and proteins.

Influenza A virus peptides NP147–155 (TYQRTRALV), NP55–69 (RLIQNSLTIERMVLS), and M2e2–24 consensus sequence (M2eCon, SLLTEVETPIRNEWGCRCNDSSD) were synthesized by the CBER core facility. The H-2d restricted adenovirus 5 hexon (Hex486–494, KYSPSNVKI) peptide (38) and a peptide pool covering B/NP of B/Ann Arbor/1/86 were synthesized by GenScript (Piscataway, NJ). A pool of severe acute respiratory syndrome coronavirus (SARS-CoV) M peptides (peptide array NR-2671) was obtained through the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH. Bacterially expressed, N-terminal His-tagged recombinant A/NP from A/PR/8/34 (H1N1) and B/NP from B/Ann Arbor/1/86 were custom produced by GenScript.

Immunologic assays.

Serum and BAL specimen antibody levels against the M2 ectodomain (M2e), A/NP, and B/NP were assessed by ELISA as described previously (14, 39). To represent the relative amount of antibody, area under the curve for absorbance versus dilution was calculated for each sample using SigmaPlot for Windows version 14.5 (Systat Software, Inc., San Jose, CA). Antigen-specific T cell responses in lungs were determined by IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay as previously described (17).

Statistical analyses.

Statistical analyses were performed using SigmaPlot for Windows versions 13 and 14.5. Antibody levels were compared by t test on log-normalized area under curve data. Virus titers and ELISPOT were analyzed by one-way analysis of variance (ANOVA) with Holm-Sidak post hoc comparison for multiple group comparisons or by t test for experiments with two groups. Transmission rate comparisons used the chi-square test or Fisher’s exact test.

ACKNOWLEDGMENTS

This work was supported by the FDA CBER Pandemic Influenza Initiative and CBER DCGT funds.

We thank Anthony Ferrine, Claudia Diaz, Mary Belcher, and staff of the FDA White Oak vivarium for outstanding support of the in vivo studies reported here and Ewan Plant and Andrew Byrnes for helpful comments during manuscript preparation.

G.E.P. and C.-Y.L. suggested the studies and designed experiments. G.E.P., C.-Y.L., and J.A.M. performed experiments and analyzed data. S.L.E., C.-Y.L., and G.E.P. wrote the manuscript. All authors edited the manuscript. S.L.E supervised the project.

Contributor Information

Graeme E. Price, Email: graeme.price@fda.hhs.gov.

Stacey Schultz-Cherry, St. Jude Children's Research Hospital.

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