Conserved HA-peptide NG34 formulated in pCMV-CTLA4-Ig reduces viral shedding in pigs after a heterosubtypic influenza virus SwH3N2 challenge

Abstract

Swine influenza viruses (SIVs), the causal agents of swine influenza, are not only important to control due to the economic losses in the swine industry, but also can be pandemic pathogens. Vaccination is one of the most relevant strategies to control and prevent influenza infection. Current human vaccines against influenza induce strain-specific immunity and annual update is required due to the virus antigenic shift phenomena. Previously, our group has reported the use of conserved hemagglutinin peptides (HA-peptides) derived from H1-influenza virus as a potential multivalent vaccine candidate. Immunization of swine with these HA-peptides elicited antibodies that recognized and neutralized heterologous influenza viruses in vitro and demonstrated strong hemagglutination-inhibiting activity. In the present work, we cloned one HA-peptide (named NG34) into a plasmid fused with cytotoxic T lymphocyte-associated antigen (CTLA4) which is a molecule that modifies T cell activation and with an adjuvant activity interfering with the adaptive immune response. The resulting plasmid, named pCMV-CTLA4-Ig-NG34, was administered twice to animals employing a needle-free delivery approach. Two studies were carried out to test the efficacy of pCMV-CTLA4-Ig-NG34 as a potential swine influenza vaccine, one in seronegative and another in seropositive pigs against SIV. The second one was aimed to evaluate whether pCMV-CTLA4-Ig-NG34 vaccination would overcome maternally derived antibodies (MDA). After immunization, all animals were intranasally challenged with an H3N2 influenza strain. A complete elimination or significant reduction in the viral shedding was observed within the first week after the challenge in the vaccinated animals from both studies. In addition, no challenged heterologous virus load was detected in the airways of vaccinated pigs. Overall, it is suggested that the pCMV-CTLA4-Ig-NG34 vaccine formulation could potentially be used as a multivalent vaccine against influenza viruses.

Fig 1. Experimental outline of two studies.

(A) 1^st study outline: seronegative pigs against SIV were vaccinated at 0 and 21 days, and challenged at 42 days. Sera were collected each pre-vaccination, pre-challenge and at necropsy day (7 dpi), as indicated. Lung tissues at 7 dpi. Nasal swabs collection took place at challenge day and at all days indicated in the figure. (B) 2^nd study outline: seropositive pigs against SIV, vaccinated at 0 and 21 days, and challenged at 42 days. Sera were collected each pre-vaccination, pre-challenge and necropsy days (7 and 14 dpi). Lung tissues were obtained at 7 and 14 dpi. In this case, bronchoalveolar fluids (BALFs) were also collected at 7 and 14 dpi. Nasal swabs collection took place at challenge day and at all days indicated in the figure. Dpi, days post-inoculation; NS, nasal swabs.

Fig 2. Representative sections from lung samples fixed with formalin and embedded in paraffin, stained with hematoxylin and eosin and microscopically evaluated following the scoring system [30] (magnification 100x): (A) scoring 0, (B) scoring 1, (C) scoring 2, and (D) scoring of 3.

Fig 3. Viral detection in nasal swabs samples by RT-qPCR.

Mean values of genomic equivalent copies (GEC) per mL obtained from nasal swabs samples (1^st study) collected at 0, 3, 5 and 7 dpi, from seronegative animals and (B) subtypic RT-qPCR results from the 1^st study (C) Mean values of GEC per mL obtained from nasal swabs samples (2^nd study) collected at 0, 1, 2, 4 and 7 dpi. from seropositive animals and (D) subtypic RT-qPCR from the 2^nd study. White bars correspond to Group A (unvaccinated group) and black bars to Group B (pCMV-CTLA4-Ig-NG34 vaccinated group). Dpi, days post-inoculation; Dashed lines indicate the detection limit of the assays: 1.24 log₁₀GEC/mL. Error bars indicate the mean ± SEM.

Fig 4. Serum antibody HA-specific IgG titers detected in sera and BALFs samples by ELISA test.

Mean of serum antibody levels detected in all individuals at time-points 0, 20 PVD and 35 PVD, and 7 dpi of Groups A and B (A) against HA from A/California/04/09(H1N1)pdm09, and (B) against HA from A/Aichi/2/1968(H3N2) are shown. Mean of BALFs antibody levels detected in pigs necropsied at 7 and 14 dpi of Groups A and B (C) against HA from A/California/04/09(H1N1)pdm09, and (D) against HA from A/Aichi/2/1968(H3N2). White circles/bars designate group A (unvaccinated group), and black squares/bars designate group B (pCMV-CTLA4-Ig-NG34 vaccinated group). OD, optical density. PVD, post-vaccination days and Dpi, days post-inoculation. Error bars indicate the mean ± SEM. Statistically significant differences between vaccine treatment groups (P value <0.05) are marked with **: p<0.01, ***: p<0.001, ****:p<0.0001.

Fig 5. HAI activity at 7 dpi against SwH3N2 in sera from seronegative pigs (Study 1).

HI titer obtained with sera from unvaccinated (Group A) and vaccinated (Group B) pigs, at 7 dpi against the SwH3N2. White circles designate group A (unvaccinated group), and black squares designate group B (pCMV-CTLA4-Ig-NG34 vaccinated group). HI: Inhibition of the hemagglutination. Error bars indicate the mean ± SEM and statistically significant differences between vaccine treatment groups (P value <0.05) are marked with *:p<0.05.

Fig 6. Influenza viral RNA detection in BALFs performed by RT-qPCR.

GEC per mL values obtained in BALFs samples, obtained from MDA positive animals (Study 2), from unvaccinated (Group A) or vaccinated (Group B) pigs at 7 dpi, corresponding to necropsy day. White circles designate group A (unvaccinated group), and black squares designate group B (pCMV-CTLA4-Ig-NG34 vaccinated group). Dashed lines indicate the detection limit of the assay: 1.24 log₁₀GEC/mL.