Matrix M H5N1 Vaccine Induces Cross-H5 Clade Humoral Immune Responses in a Randomized Clinical Trial and Provides Protection from Highly Pathogenic Influenza Challenge in Ferrets

Abstract

Background and methods: Highly pathogenic avian influenza (HPAI) viruses constitute a pandemic threat and the development of effective vaccines is a global priority. Sixty adults were recruited into a randomized clinical trial and were intramuscularly immunized with two virosomal vaccine H5N1 (NIBRG-14) doses (21 days apart) of 30 μg HA alone or 1.5, 7.5 or 30 μg HA adjuvanted with Matrix M. The kinetics and longevity of the serological responses against NIBRG-14 were determined by haemagglutination inhibition (HI), single radial haemolysis (SRH), microneutralization (MN) and ELISA assays. The cross-H5 clade responses in sera were determined by HI and the antibody-secreting (ASC) cell ELISPOT assays. The protective efficacy of the vaccine against homologous HPAI challenge was evaluated in ferrets.

Results: The serological responses against the homologous and cross-reactive strains generally peaked one week after the second dose, and formulation with Matrix M augmented the responses. The NIBRG-14-specific seroprotection rates fell significantly by six months and were low against cross-reactive strains although the adjuvant appeared to prolong the longevity of the protective responses in some subjects. By 12 months post-vaccination, nearly all vaccinees had NIBRG-14-specific antibody titres below the protective thresholds. The Matrix M adjuvant was shown to greatly improve ASC and serum IgG responses following vaccination. In a HPAI ferret challenge model, the vaccine protected the animals from febrile responses, severe weight loss and local and systemic spread of the virus.

Conclusion: Our findings show that the Matrix M-adjuvanted virosomal H5N1 vaccine is a promising pre-pandemic vaccine candidate.

Trial registration: ClinicalTrials.gov NCT00868218.

Conflict of interest statement

Competing Interests: The authors have the following interests. Crucell provided the vaccine for this study. There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Fig 1. CONSORT flowchart.

In total, 65 adult subjects were assessed for eligibility and 60 were included in the study (mean age 31 years old, 60% female). Prior to being enrolled in the study, all participants provided written informed consent and were screened for normal haematology, biochemistry and immunology parameters. The 60 volunteers were randomised into four groups and were immunised twice, 21 days (±1) apart with the virosomal H5N1 vaccine alone (30μg HA) or with vaccine (1.5, 7.5 or 30μg HA) formulated with Matrix-M (50μg) adjuvant. Blood samples were collected at 7, 14 and 21 days after each dose of vaccine and 180 and 365 days after the first vaccination.

Fig 2. The kinetics and long-term microneutralization response after vaccination.

The kinetics (A) and long-term (B) microneutralization (MN) antibody response to the homologous vaccine strain A/Vietnam/1194/2004 (NIBRG-14) after vaccination with two doses (21 ±1 days apart) of inactivated virosomal H5N1 vaccine alone (30μg HA, blue) or 1.5 (red), 7.5 (green) or 30μg HA (black) adjuvanted with Matrix-M (50μg). The sampling day after vaccination is shown on the x-axis. Each symbol represents the geometric mean MN titre for one individual participant, with the group geometric mean titre and 95% confidence interval presented. The dotted line indicates the protective MN titre of 80.

Fig 3. The kinetics and long-term single radial haemolysis antibody response induced after vaccination.

The kinetics (A) and long-term (B) single radial haemolysis response to the homologous vaccine strain A/Vietnam/1194/2004 (NIBRG-14) after vaccination with two doses (21 ±1 days apart) of inactivated virosomal H5N1 vaccine alone (30μg HA, blue) or 1.5 (red), 7.5 (green) or 30 μg HA (black) adjuvanted with Matrix-M (50μg). The sampling day after vaccination is shown on the x-axis. Each symbol represents the geometric mean lysis zone area (mm2) for one individual participant, with the group geometric mean and 95% confidence interval presented. The dotted line shows the seroprotective SRH Zone area of ≥25mm2.

Fig 4. The kinetics of the haemagglutination inhibition antibody response induced after vaccination.

The kinetics of the haemagglutination inhibition (HI) antibody response to the homologous vaccine strain (A) A/Vietnam/1194/2004 (NIBRG-14) and the cross-H5 clade HI responses to (B) A/Cambodia/R0405050/2007 (NIBRG-88), (C) A/Indonesia/5/2005 (RG2) and (D) A/turkey/Turkey/1/2005 (NIBRG-23) strains were measured by the HI assay. Subjects were vaccinated with two doses (21 ±1 days apart) of inactivated virosomal H5N1 vaccine alone (30μg HA, blue) or 1.5 (red), 7.5 (green) or 30μg HA (black) adjuvanted with Matrix-M (50μg). The sampling day after vaccination is shown on the x-axis. The HI titres were measured by the modified HI test using horse erythrocytes. Each symbol represents the geometric HI titre for one individual, with the group geometric mean and 95% confidence interval presented. The dotted line shows the protective HI titre of 32.

Fig 5. The longevity of the haemagglutination inhibition antibody response induced after vaccination.

A: The haemagglutination inhibition (HI) response against the homologous vaccine strain A/Vietnam/1194/2004 (NIBRG-14) at 180 and 365 days after vaccination. B: The cross-H5 clade HI response to A/turkey/Turkey/1/2005 (NIBRG-23), A/Indonesia/5/2005 (RG2) and A/Cambodia/R0405050/2007 (NIBRG-88) at 180 days post-vaccination. Subjects were vaccinated with two doses (21 ±1 days apart) of inactivated virosomal H5N1 vaccine alone (30μg HA, blue) or 1.5 (red), 7.5 (green) or 30 μg HA (black) adjuvanted with Matrix-M (50μg). The HI titres were measured by the modified HI test using horse erythrocytes. Each symbol represents the geometric HI titre for one individual participant, with geometric mean titres for the group and 95% confidence interval presented. The dotted line shows the protective HI titre of 32.

Fig 6. The H5N1-specific IgG response after vaccination.

An ELISA was used to measure the NIBRG-14 influenza vaccine-specific serum IgG response before and after vaccination. Subjects were vaccinated with two doses (21 ±1 days apart) of inactivated virosomal H5N1 vaccine alone (30μg HA, blue) or 1.5 (red), 7.5 (green) or 30μg HA (black) adjuvanted with Matrix-M (50μg). The bars represent the mean IgG titre ± standard error of the mean.

Fig 7. Correlation between serological responses to the homologous and cross-reactive H5N1 strains at days 21, 42 and 180 after vaccination.

The correlation between A/Vietnam/1194/2004 (NIBRG-14)-specific HI, SRH, MN and IgG responses and the HI responses against A/turkey/Turkey/1/2005 (NIBRG-23), A/Cambodia/R0405050/2007 (NIBRG-88) and A/Indonesia/5/2005 (RG2) at 21, 42 and 180 days after vaccination. Volunteers were divided into four vaccine groups and were vaccinated with two doses (21 ±1 days apart) of inactivated virosomal H5N1 vaccine alone (30μg HA) or 1.5, 7.5 or 30μg HA adjuvanted with Matrix-M (50μg). Data from the four vaccine groups were combined to calculate the Spearman rank correlation coefficient (r) value (adjusted for multiple comparisons) for each association between the homologous and cross-H5 clade serological response. Abbreviations: p, Two-tailed p value (95% confidence interval); NS, no significant correlation.

Fig 8. The antibody secreting cell response induced after influenza vaccination.

Lymphocytes were collected 7 days after the first (day 7) and second vaccine dose (day 28) of inactivated virosomal H5N1 vaccine alone or formulated 1.5, 7.5 or 30μg HA formulated with Matrix-M (50μg). The influenza-specific IgG, IgA and IgM antibody secreting cells (ASC) were enumerated using the ELISPOT assay. The data are presented as the mean number of influenza-specific ASC per 200 000 lymphocytes ± standard error of the mean to the homologous vaccine strain A/Vietnam/1104/2004 (NIBRG-14), and the heterologous responses to A/Turkey/Turkey/1/05 (NIBRG-23), A/Cambodia/R0405050/2007 (NIBRG-88) and A/Anhui/1/05 (RG6). Statistical differences between the adjuvanted and non-adjuvanted groups were calculated by ANOVA with Dunnett’s multiple comparisons test. *p

Fig 9. The memory B cell (Bmem) response 365 days after vaccination.

Subjects were vaccinated with two doses (21 ±1 days apart) of inactivated virosomal H5N1 vaccine alone (30μg HA, blue) or 1.5 (red), 7.5 (green) or 30μg HA (black) adjuvanted with Matrix-M (50μg). The Bmem response at 365 days post-first vaccination was analyzed by ELISPOT. The bars represent the mean number of NIBRG-14-specific Bmem per 1×106 peripheral blood mononuclear cells (PBMC) ± standard error of the mean.

Fig 10. Protective efficacy of the H5N1 vaccine in ferrets.

Ferrets were divided into five groups of 10 and were vaccinated intramuscularly with two doses (14 days apart) of virosomal vaccine alone (30μg HA, blue) or 1.5 (red), 7.5 (green) and 30μg HA (black) adjuvanted with Matrix M (50μg). Control ferrets (brown) received PBS. Fourteen days after the second immunization, ferrets were challenged by administering A/Vietnam/1203/2004 live virus (106 EID50 in 0.4 mL PBS/BSA). (A) The serum HI antibody response 12 days after each vaccination with NIBRG-14 virosomal vaccine and fourteen days following challenge with A/Vietnam/1203/2004 virus. (B) The virus titres in the nasal washing and tissue samples after challenge with the A/Vietnam/1203/2004 virus. The limit of detection in TCID50 assay for the ferret tissue samples was 101.63TCID50/ml of homogenate. (C) The changes in weight and (D) temperature after challenge with A/Vietnam/1203/2004 live virus.