Unconjugated Multi-Epitope Peptides Adjuvanted with ALFQ Induce Durable and Broadly Reactive Antibodies to Human and Avian Influenza Viruses

Abstract

An unconjugated composite peptide vaccine targeting multiple conserved influenza epitopes from hemagglutinin, neuraminidase, and matrix protein and formulated with a safe and highly potent adjuvant, Army Liposome formulation (ALFQ), generated broad and durable immune responses in outbred mice. The antibodies recognized specific epitopes in influenza peptides and several human, avian, and swine influenza viruses. Comparable antibody responses to influenza viruses were observed with intramuscular and intradermal routes of vaccine administration. The peptide vaccine induced cross-reactive antibodies that recognized influenza virus subtypes A/H1N1, A/H3N2, A/H5N1, B/Victoria, and B/Yamagata. In addition, immune sera neutralized seasonal and pandemic influenza strains (Group 1 and Group 2). This composite multi-epitope peptide vaccine, formulated with ALFQ and administered via intramuscular and intradermal routes, provides a high-performance supra-seasonal vaccine that would be cost-effective and easily scalable, thus moving us closer to a viable strategy for a universal influenza vaccine and pandemic preparedness.

Keywords: ALFQ; QS21 (also known as QS-21); broadly reactive antibodies; durable; immune responses; influenza; intradermal; intramuscular; liposomes; neutralizing antibodies; peptides; universal vaccine.

Figure 1

Serum IgG1 responses to the composite peptides HA + NA and M1/M2/M2e + T-cell epitope in mice immunized with either 1 µg or 20 µg of LHNVD-105 formulated with ALFQ, administered intramuscularly or intradermally. Day 0, 21, 35, 42, 56 and 63 sera samples were analyzed using ELISA. IgG1 titers to HA + NA (Flu Pep11) in intramuscular (A) and intradermal (B) groups; M1/M2/M2e + T-cell epitope (Flu Pep5906) titers in intramuscular (C) and in intradermal (D). Data are expressed as mean ± SD. * p = 0.01, ** p = 0.001, **** p < 0.0001.

Figure 2

Serum IgG2b responses to the composite peptides HA + NA and M1/M2/M2e + T-cell epitope in mice immunized with either 1 µg or 20 µg of LHNVD-105 formulated with ALFQ, administered intramuscularly or intradermally. Day 0, 21, 35, 42, 56 and 63 sera samples were analyzed using ELISA. IgG2b titers to HA + NA (Flu Pep11) in intramuscular (A) and intradermal (B) groups; M1/M2/M2e + T-cell epitope (Flu Pep5906) titers in intramuscular (C) and intradermal (D). Data are expressed as mean ± SD. ** p = 0.001, **** p < 0.0001.

Figure 3

Serum IgG1 and IgG2b responses to the composite peptides HA + NA and M1/M2/M2e + T-cell epitope in mice immunized with either 1 µg or 20 µg of LHNVD-105 with ALFQ, administered intramuscularly or intradermally. Longevity of titers in sera samples up to day 200 was determined using ELISA. (A,C) demonstrate IgG1 and IgG2b titers to HA + NA (Flu Pep11) and (B,D), to M1/M2/M2e + T-cell epitope (Flu Pep5906), respectively. Data are expressed as mean ± SD.

Figure 4

Antisera responses, at day 56, to individual HA, NA and M2e epitopes in mice immunized with LHNVD-105, formulated with ALFQ, administered either intramuscularly or intradermally at two doses of 1 µg of 20 µg. IgG1 titers to individual HA, NA epitopes and the composite peptide HA + NA are shown in panel (A) and titers to M1, M2 epitopes and the full-length M1/M2/M2e are shown in panel (B). Data from each group (5 mice/group) are expressed as mean ± SD. Statistical significance between titers to individual peptides for each group was calculated using the two-way ANOVA method with Šidák correction for multiple comparison, and the significance threshold set at p < 0.05 (* p = 0.01). Pre-immunization pooled serum (control) had absorbance values of 0.094–0.104 on different antigens. Endpoint titers are the reciprocal of highest dilution that yielded absorbance values greater than or equal to twice that of the pooled pre-immune values.

Figure 5

Isotype-specific IgG antisera responses, at day 49 and day 154, in mice immunized with LHNVD-105 with adjuvant ALFQ. IgG2a titers to HA + NA and M1/M2/M2e + T-cell epitope at day 49 and day 154 are shown in panels (A,B), and IgG3 titers at day 49 and day 154 are demonstrated in panels (C,D), respectively. Data from each group (5 mice/group) are expressed as mean ± SD. Pre-immunization pooled serum (control) had absorbance values of 0.053–0.076 with different IgG isotypes. Endpoint titers are the reciprocal of highest dilution that yielded absorbance values greater than or equal to twice that of the pooled pre-immune values. Dashed line at 100 represents minimum calculated titer. Titers below 100 are reported as nil.

Figure 6

Isotype-specific IgG antisera responses, at day 49, in mice immunized with LHNVD-105 with ALFQ adjuvant. IgG1 and IgG2a titers to HA + NA and M1/M2/M2e + T-cell epitope at day 49 are shown in panels (A,B), respectively. Data from each group (5 mice/group) are expressed as mean ± SD. Pre-immunization pooled serum (control) had absorbance values of 0.053–0.129 with different IgG isotypes. Endpoint titers are the reciprocal of highest dilution that yielded absorbance values greater than or equal to twice that of the pooled pre-immune values.

Figure 7

Day 49 (A,C) and day 154 (B,D) pooled sera from mice immunized with LHNVD-105 formulated with ALFQ, at two doses of 1 and 20 µg administered intramuscularly or intradermally, respectively, recognized live/inactivated contemporary influenza A virus of subtypes H1N1, H3N2, H5N1 and live influenza B virus of Yamagata lineage. IgG1 titers to viruses in pooled sera from each group were analyzed using ELISA. Data expressed as mean ± SD from three separate experiments with pooled sera from each group. Pre-immunization pooled serum (control) had mean absorbance values of 0.110–0.144 on different virus strains. Endpoint titers are the reciprocal of highest dilution that yielded absorbance values greater than or equal to twice that of the pooled pre-immune values.

Figure 8

Day 200 pooled sera from mice immunized with LHNVD-105 formulated with ALFQ, at two doses of 1 and 20 µg administered intramuscularly or intradermally, respectively, recognized contemporary live influenza A virus of subtypes H1N1 and H3N2 (A), inactivated H5N1 (B) and live influenza B virus of Victoria and Yamagata lineages (C). IgG1 titers to viruses in pooled sera from each group were analyzed using ELISA. Data expressed as mean ± SD from three separate experiments with pooled sera from each group. Pre-immunization pooled serum (control) had mean absorbance values of 0.074–0.096 on different virus strains. Endpoint titers are the reciprocal of highest dilution that yielded absorbance values greater than or equal to twice that of the pooled pre-immune values. No statistical significance was observed between the groups (p > 0.05).

Figure 9

Day 42 to Day 63 serum samples from mice immunized with LHNVD-105 with adjuvant ALFQ neutralized live contemporary influenza A virus of H1N1 (A) and H3N2 (B) subtypes. Pooled sera from each group were analyzed for virus neutralization using MNA. Titer represents neutralization of the virus in three out of four wells. Negative control titer was at 10¹. Data from three (triangles in panel A) to five (circles in panel B) separate experiments are expressed as mean ± SD.