Influenza Antigens NP and M2 Confer Cross Protection to BALB/c Mice against Lethal Challenge with H1N1, Pandemic H1N1 or H5N1 Influenza A Viruses

Abstract

Influenza hemagglutinin (HA) is considered a major protective antigen of seasonal influenza vaccine but antigenic drift of HA necessitates annual immunizations using new circulating HA versions. Low variation found within conserved non-HA influenza virus (INFV) antigens may maintain protection with less frequent immunizations. Conserved antigens of influenza A virus (INFV A) that can generate cross protection against multiple INFV strains were evaluated in BALB/c mice using modified Vaccinia virus Ankara (MVA)-vectored vaccines that expressed INFV A antigens hemagglutinin (HA), matrix protein 1 (M1), nucleoprotein (NP), matrix protein 2 (M2), repeats of the external portion of M2 (M2e) or as tandem repeats (METR), and M2e with transmembrane region and cytoplasmic loop (M2eTML). Protection by combinations of non-HA antigens was equivalent to that of subtype-matched HA. Combinations of NP and forms of M2e generated serum antibody responses and protected mice against lethal INFV A challenge using PR8, pandemic H1N1 A/Mexico/4108/2009 (pH1N1) or H5N1 A/Vietnam/1203/2004 (H5N1) viruses, as demonstrated by reduced lung viral burden and protection against weight loss. The highest levels of protection were obtained with NP and M2e antigens delivered as MVA inserts, resulting in broadly protective immunity in mice and enhancement of previous natural immunity to INFV A.

Keywords: conserved antigens; hemagglutinin; influenza A; matrix protein 1; matrix protein 2; modified Vaccinia virus Ankara (MVA); nucleoprotein; vaccine.

Figure 1

Influenza matrix protein M2 structural features and comparison to METRC used in this study. M2e domains (red in diagram) are the external 23 amino acids of the M2 molecule. An M2e TML construct contained M2e from PR8 H1N1 and a conserved transmembrane region with an additional 16 cytoplasmic amino acids for a total of 58 amino acids. The M2 molecule in nature consists of an external domain (red, M2e), a transmembrane domain (slashes, grey), and a cytoplasmic domain (lined, green) for a total of 97 amino acids. METRC contains 6 versions of M2e domains (red), each found in nature. Amino acid sequences of each version of M2e found in METRC are listed with amino acid substitutions relative to peptide #3 which is native to PR8.

Figure 2

Body weight loss after PR8 H1N1 challenge of mice vaccinated with MVA-based vaccines. Combined conserved non-HA antigen MVA vaccines resulted in weight loss similar to an HA MVA vaccine. Mice were vaccinated intramuscularly two times, days 0 and 18–22, and challenged intranasally on day 42. (A) HA was superior to other single non-HA antigens. (B) Combined antigen vaccines resulted in less than 7% weight loss. (C) Data for NP and M2 vaccines, alone and combined. (D) Data for NP and M2eTML vaccines, alone and combined. (E) Data for NP and METRC vaccines, alone and combined. (F) Data for NP and METRS vaccines, alone and combined.

Figure 3

Change in body weight and survival of BALB/c mice challenged with six LD50 (LD99) pH1N1. Eight groups of mice (N = 8) either immunized with various MVA-vector based vaccines or with MVA vector (MVAtor) were challenged with pH1N1 3 weeks after the final immunization. Daily body weights were monitored up to 14 days post-challenge. Weight loss >20% body weight was scored as death. Vaccines that delivered NP combined (blended) with M2 or METRC had similar (15%) weight loss that improved over the monitored period. Despite substantial weight loss in recipients of single antigen MVA-M1 vaccine, M1 did not lessen the benefit of blended MVA M1 + MVA NP + MVA M2 or blended MVA M1 + MVA NP + MVA METRC vaccines.

Figure 4

Based upon body weight of BALB/c mice after H5N1 challenge, non-HA antigen vaccines can build upon immunity conferred previously by natural heterologous infection (empty squares), reaching the level of weight loss protection provided by H5 HA (solid red diamonds) delivered two times. Mice in groups of eight were immunized two times using MVA vaccines (solid symbols) or after natural INFV exposure (“Prime”, empty symbols) using low dose non-lethal PR8 INFV A. After H5N1 lethal challenge, mice were monitored for daily body weight and survival for 14 days. The period of 4 to 7–8 days post challenge is framed in each panel to illustrate absence of weight loss due to natural INFV exposure. Heterologous exposure to native PR8 H1N1 INFV A, followed by administration of NP + METRC + M1 expressed as a single insert in MVA ((A), empty squares, “Prime, single insert”) or as a blended vaccine of individually expressed antigens in MVA ((B), empty squares, “prime, blend”) conferred minor weight loss similar to homologous HA vaccine.

Figure 5

Lung viral burdens after immunization and H5N1 challenge. Mice were immunized two times using a single MVA vaccine that encoded M1 + NP + METRC (“single insert”) or a combination of three separate MVA vaccines each encoding M1 or NP or METRC (“Blend”). “Prime” indicates previous mild infection with PR8 H1N1 INFV A, 21 days prior to vaccine administration. A TCID₅₀ assay was performed to quantify infectious H5N1 virus in lungs (N = 5 per group) 4 days after A/VN/1203/04 challenge (H5N1). The horizontal bar for each group represents the arithmetic mean Log₁₀ TCID₅₀ per gram tissue. Individual dots represent the results of each animal. The limit of detection for this assay was 1.7 Log₁₀ TCID₅₀. Lung viral burden on day 2 (data not shown) had nearly identical results compared to day 4. Without PR8 priming, only the MVA H5HA vaccine was different (p < 0.0001, ANOVA followed by Tukey–Kramer test) from the MVAtor control. If delivered after PR8 priming, MVA encoding M1 + NP + METRC as a single insert again was significantly different from MVAtor with priming (p = 0.019). Priming gave protective advantage to all test vaccines including the MVAtor negative control. Limit of detection was log 1.7.

Figure 6

Levels of anti-NP IgG in sera of mice after vaccination with MVA vaccines that encoded NP combined with other antigens. Levels of serum anti-NP IgG were analyzed by ELISA. “Prime” refers to intranasal instillation of sublethal PR8 on day 0; first immunization using an MVA vaccine occurred on day 22; second immunization using MVA vaccines occurred on day 43. MVAtor alone did not result in serum anti-NP IgG antibody levels but priming using PR8 generated high IgG anti-NP levels. Squares depict single insert encoding M1 + METRC + NP; triangles depict a blend of individual M1, METRC and NP constructs; circles depict MVAtor controls. Filled symbols depict MVA vaccine treatments; empty symbols depict non-lethal (10% weight loss) PR8 infection as a priming event. * Anti-NP IgG ELISA were not performed using sera of this group.

Figure 7

Anti-M2 IgG immune response in mice that received vaccines that encoded METRC antigen (Immunization days: Optional prime day 0, MVA Day 22 and Day 43). “PR8 Prime” refers to intranasal installation of sublethal PR8 on day 0; first immunization using an MVA vaccine occurred on day 22; second immunization using MVA vaccines occurred on day 43. For virus-primed mice, serum anti-M2 IgG antibody levels did not increase beyond the primed and boosted level despite an additional immunization. Squares depict single insert encoding M1 + METRC + NP; triangles depict a blend of individual M1, METRC and NP constructs; circles depict MVAtor controls. Filled symbols depict MVA vaccine treatments; empty symbols depict non-lethal PR8 infection as a priming event.