Live attenuated influenza virus vaccine reduces virus shedding of newborn piglets in the presence of maternal antibody

Background Influenza A virus in swine (IAV‐S) causes an acute respiratory disease of swine which results in great economic losses in pig production. Major control strategies include the use of killed vaccines (KV) in breeding females to confer passive immunity to their offspring. A bivalent H1N1 and H3N2 NS1‐truncated live attenuated IAV‐S vaccine have recently become available, which showed promising results in young pigs. Objective The aim of this study was to investigate the effect of an intranasal vaccination of newborn pigs with or without maternally derived antibodies (MDA) on virus shedding (via nasal swabs tested by virus isolation). Methods The study was performed as intratracheal challenge experiments with either a heterologous H1N2 or H3N2 viruses. Results and conclusion The results of this study showed a significant decrease in the incidence and duration of shedding viable virus for vaccinated newborn piglets with or without MDA, providing strong evidence that intranasal vaccination is overcoming passively acquired maternal immunity. This study indicates that intranasal vaccination with a truncated NS1 live attenuated IAV‐S vaccine of newborn piglets with maternal antibodies can be a valuable tool for reducing the prevalence of heterologous H1N2 and H3N2 IAV‐S in pig herds.

shedding (via nasal swabs tested by virus isolation).

Methods:
The study was performed as intratracheal challenge experiments with either a heterologous H1N2 or H3N2 viruses.

Results and conclusion:
The results of this study showed a significant decrease in the incidence and duration of shedding viable virus for vaccinated newborn piglets with or without MDA, providing strong evidence that intranasal vaccination is overcoming passively acquired maternal immunity. This study indicates that intranasal vaccination with a truncated NS1 live attenuated IAV-S vaccine of newborn piglets with maternal antibodies can be a valuable tool for reducing the prevalence of heterologous H1N2 and H3N2 IAV-S in pig herds. used primarily in breeding females to confer passive immunity to their offspring, which does not protect piglets against infection and transmission of IAV-S but rather against clinical disease). 1 Recently, a live attenuated influenza virus vaccine (LAIV) has been reported to be efficacious in heterologous challenges in young piglets. 2 Modifications introduced into the viral NS1 gene via reverse genetics have resulted in modified live influenza viruses that have been shown to be efficacious. 3,4 The aim of this study was to investigate whether vaccination with this LAIV would reduce viral shedding (duration and incidence) in newborn piglets with or without maternally derived antibodies (MDA).

| Vaccines and challenge virus
The IAV-S LAIV was formulated by combining two viruses, the previously described I-A triple-reassortant internal gene (TRIG) cluster H3N2 5 and a novel α-cluster H1N1 which was constructed using the same techniques. Once combined, the viruses were lyophilized to create a bivalent vaccine. The vaccine was administered at a dose of 1 mL intranasally to one nostril.

| Experimental design
All study procedures and animal care activities were conducted at the ratios using a cutoff of S/N <0.60 as positive. S/N results of dams range from 0.336 to 0.083. Sows were randomized to one of two rooms, and then, the treatment (control or vaccine) was randomized to room. As an additional challenge control source of piglets, seronegative sows were also obtained and housed separately from the seropositive sows (Wilson Prairie View Farms, Burlington, WI, USA).
Ten dams tested negative for IAV NP by MultiS-Screen Ab ELISA at ISU-VDL and were randomized to one of two additional rooms, and all study activities occurred in parallel with the seropositive group. Three vaccinated pigs and four or five seropositive control pigs were commingled into a single pen (as litter sizes were uneven, some litters were split between pens). For each study time point, the same design (in a separate room) was applied to seronegative pigs with two pigs per litter randomized to the H1@4 and H3@5 challenge studies and the remainder included in H1@9 challenge study (Table 1).

| Observations and sampling
Pigs were monitored daily for general demeanor, respiratory signs, and rectal temperature from −2 to 5 days post-challenge (dpc). Nasal swabs were collected daily from −1 to 5 dpc by insertion of a single swab (Fisherbrand, poly tip Catalog No. 23-400-122) into each nostril.
The swabs were then stored frozen at −70 °C in a 5-mL tube containing 2 mL of tissue culture media formulated with antibiotic and antimycotic until testing could be completed. The plates were sealed and incubated at room temperature for approximately 1 hour or until the negative control wells formed a button and the positive control wells formed a mat.

| Statistics
The

| NP ELISA
Prior to treatment, all pigs were tested to confirm their serostatus (positive or negative) by the NP ELISA ( Table 2). The serostatus was investigated again prior to either challenge. There was a decline in titers in all seropositive groups as evidence of a decay of maternal antibodies. The results also show that following intranasal vaccination, there was little to no seroconversion in all three experiments.

| Respiratory signs and body temperature
Pigs showed a notable increase in respiratory effort 2 days after challenge in all groups; however, the control groups had more pigs with severe dyspnea than the vaccinated groups. Respiratory signs lasted for not longer than 4 dpc in all groups (data not shown).
All three challenge events (H1@4, H3@5, and H1@9) resulted in a significant increase in body temperature compared to baseline for all treatment groups 1-day post-challenge (P ≤ .05) for both seropositive and seronegative animals ( Figure 1A,

| Virus isolation of nasal swabs
All pre-challenge nasal swabs of all groups tested negative for IAV-S. Animals in the control group had more virus-positive nasal swabs compared to vaccinated groups, which was significant on 4 and 5 dpc in H1@4w ( Figure 2A) and H1@9w ( Figure 2C) and significant on 3, 4, and 5 dpc in H3@5w (P ≤ .05) ( Figure 2B).

| Median duration shedding over time
As evident from Figure  were made between challenge studies as two different isolates were used in this study and pathogenicity of each virus was not established prior to study initiation.
A recent study has shown that piglets play a key role in maintaining IAV-S in the breeding herds, 11 which provides evidence that early piglet vaccination with a vaccine that provides heterologous protection will contribute to reduce IAV-S prevalence both in a herd and between herds. More studies are needed to quantify the reduction in prevalence by including more systems of different sizes and management practices.
In conclusion, this study provides evidence that intranasal vaccination of MDA-positive piglets with a truncated NS1 live attenuated influenza A vaccine resulted in significantly reduced viral replication and shedding after both H1N2 and H3N2 challenges.