Research ArticleVACCINES

Nucleoside-modified mRNA encoding HSV-2 glycoproteins C, D, and E prevents clinical and subclinical genital herpes

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Science Immunology  20 Sep 2019:
Vol. 4, Issue 39, eaaw7083
DOI: 10.1126/sciimmunol.aaw7083
  • Fig. 1 ID immunization with nucleoside-modified trivalent mRNA-LNP protects against genital HSV-2 in mice.

    (A to F) BALB/c mice were immunized twice with PolyC RNA-LNP, gD2 nucleoside-modified mRNA-LNP, and trivalent nucleoside-modified mRNA-LNP or three times with trivalent subunit protein–CpG/alum and were intravaginally challenged 1 month after the final immunization with 5 × 103 PFU of HSV-2. (A) Survival curves: P < 0.001 comparing PolyC RNA group with each other. P values were calculated by the log-rank test. CI, confidence interval. (B) The mean weight loss each day after infection of animals in the trivalent mRNA, trivalent subunit protein, or gD2 mRNA group was compared with PolyC RNA controls; P < 0.0001. (C) Mean genital disease scores after infection comparing trivalent mRNA, trivalent subunit protein, or gD2 mRNA with PolyC RNA; P < 0.0001. P values in (B) and (C) were calculated by two-way ANOVA, followed by Tukey for significance. Error bars in (C) represent SEM. (D) Day 2 and 4 vaginal titers. Dotted line indicates the limit of assay detection at 6.7 PFU/ml. GMT, geometric mean titer. (E) Day 2 and 4 vaginal DNA copy number by qPCR. (F) Day 4 DRG HSV-2 DNA copy number by qPCR. P values in (D) to (F) are shown in the figures for the primary end point comparison between trivalent mRNA and trivalent protein or secondary end point comparison between trivalent mRNA and gD2 mRNA and were calculated by the two-tailed Fisher’s exact test. Sample size: (A to C and F) n = 10 per group, except trivalent mRNA group, n = 20 for (A) to (C) and n = 19 for (F); (D) n = 10 in PolyC RNA, n = 20 in gD2 mRNA, n = 39 in trivalent mRNA, and n = 10 in trivalent subunit protein; (E) n = 10 per group, except trivalent mRNA group, n = 20. (G) Challenge of trivalent mRNA–immunized mice with 5 × 104 PFU (2750 LD50).

  • Fig. 2 IM immunization with nucleoside-modified trivalent mRNA-LNP protects against genital HSV-2 in mice.

    BALB/c mice were immunized as in Fig. 1, except that one group received 2 μg of trivalent mRNA. Mice were intravaginally challenged 1 month after the final immunization with 5 × 103 PFU of HSV-2 (275 LD50). (A) Survival curves: P < 0.0001 comparing PolyC RNA group with each other. P values were calculated by the log-rank test. (B) Weight loss of individual animals: P < 0.0001 comparing PolyC RNA with the other three groups. (C) Genital disease: P < 0.0001 comparing PolyC RNA with the other three groups. P values in (B) and (C) were calculated by two-way ANOVA followed by Tukey for significance. Sample size: (A to C), n = 10 in the PolyC RNA group for (A) and (C), n = 5 for (B), n = 10 in the trivalent subunit protein group, n = 5 in the 2 μg trivalent mRNA group, and n = 10 in the 10 μg trivalent mRNA group. Error bars in (C) represent SEM. P values in (D) and (E) are shown on the figures for the primary end point comparison between trivalent mRNA and trivalent protein or the comparison between 2 and 10 μg of trivalent mRNA. Other P values are noted below. (D) Day 2 vaginal titers: P < 0.0001, comparing 10 μg of trivalent mRNA or trivalent subunit protein with PolyC RNA; P = 0.2500, comparing 2 μg of trivalent mRNA with PolyC RNA. Day 4 vaginal titers: P < 0.0001, comparing the number of animals with positive day 4 cultures in the 10 μg trivalent mRNA group, 2 μg trivalent mRNA group, or trivalent subunit protein group with PolyC RNA. Sample size: n = 15 in the PolyC RNA group, n = 10 in the trivalent subunit protein group, n = 5 in the 2 μg trivalent mRNA group, and n = 20 in the 10 μg trivalent mRNA group. Dotted line indicates the limit of assay detection at 6.7 PFU/ml. (E) DRG HSV-2 DNA copy number at time of euthanasia of PolyC RNA group or on day 28 in the other groups; P < 0.0001, comparing the number of animals with positive HSV-2 DNA copy number in the PolyC RNA with the 10 μg trivalent mRNA group or the trivalent subunit protein group; P = 0.0050, comparing the number of animals with positive HSV-2 DNA copy in the PolyC RNA with the 2 μg trivalent mRNA group. Other comparisons were not significant. Sample size: n = 9 in the PolyC RNA group, n = 10 in the trivalent subunit protein group, n = 5 in the 2 μg trivalent mRNA group, and n = 10 in the 10 μg trivalent mRNA group. P values in (D) and (E) were calculated by the two-tailed Fisher’s exact test.

  • Fig. 3 ID immunization with nucleoside-modified trivalent mRNA-LNP protects against genital HSV-2 in guinea pigs.

    (A to F) Guinea pigs were immunized three times monthly IM with 10 μg of PolyC RNA-LNP, ID with 20 μg of each trivalent nucleoside-modified mRNA-LNP, and IM with 10 μg of each trivalent subunit protein–CpG/alum or left unimmunized (n = 10 per group). One month after the final immunization, all immunized animals were infected intravaginally with 5 × 105 PFU of HSV-2 (50 LD50), whereas unimmunized animals remained uninfected. (A) Survival curves: P < 0.0001, comparing PolyC RNA group with each other. P values calculated by the log-rank test. (B) The mean weight loss each day after infection of animals in the trivalent mRNA, trivalent subunit protein, or uninfected group was compared with PolyC RNA controls; P < 0.0001. (C) Cumulative mean genital disease score per guinea pig is shown for each day after infection. No animal developed genital lesions in the trivalent mRNA, trivalent subunit protein, or uninfected group. P < 0.0001, comparing trivalent mRNA, trivalent subunit protein, or uninfected with PolyC RNA. The P values in (B) and (C) were calculated by two-way ANOVA followed by Tukey for significance. (D and E) P values shown in the figures are for the primary end point comparison between trivalent mRNA and trivalent protein. Other P values are noted below. (D) Day 2 and 4 vaginal titers: P = 0.0013 (day 2) and P = 0.0010 (day 4), comparing PolyC RNA with trivalent mRNA. Dotted line indicates the limit of assay detection at 3.3 PFU/ml. (E) Day 2 and 4 vaginal DNA copy number by qPCR. P values in (D) and (E) were calculated by the two-tailed Mann-Whitney, except that the day 4 comparison between trivalent mRNA and trivalent subunit proteins was performed by the two-tailed Fisher’s exact test. (F) Vaginal shedding of HSV-2 DNA over 21 days from days 28 to 50 after infection. One animal in the trivalent subunit protein group had replication-competent virus recovered on the day HSV-2 DNA shedding was detected (marked in green). P = 0.0052 (calculated by the two-tailed Fisher’s exact test), comparing days of shedding in the trivalent mRNA and trivalent subunit protein groups. (G) HSV-2 DNA copy number per 106 copies of GAPDH in spinal cord and DRG at the end of the experiment; n = 2 in the PolyC RNA group and n = 10 in the 3 other groups. (H) Table summarizing key results of guinea pig studies.

  • Fig. 4 ELISA, neutralizing antibody, and ADCC responses.

    Mice were immunized ID with PolyC RNA-LNP, gD2 mRNA-LNP, and 10 μg of trivalent mRNA-LNP or IM with trivalent subunit protein–CpG/alum. Sera were obtained 4 weeks after the first and second immunizations in the mRNA-LNP groups and 4 weeks after the third immunization in the subunit protein group. P values are shown on the figures for the primary end point comparison between trivalent mRNA and trivalent protein or secondary end point comparison between trivalent mRNA and gD2 mRNA. Other P values are noted below. (A) Serum gC2 IgG: P = 0.0058, comparing first and second trivalent mRNA immunizations. (B) Serum gD2 IgG: P = 0.6060, comparing final immunizations of trivalent mRNA with gD2 mRNA; P = 0.0085, comparing first and second gD2 mRNA immunizations; P = 0.0056, comparing first and second trivalent mRNA immunizations. (C) Serum gE2 IgG: P = 0.0058, comparing first and second trivalent mRNA immunizations. n = 9 animals per group. (A to C) P values comparing the first immunization with the second immunization were calculated by the two-tailed Wilcoxon signed-rank test. P values comparing final immunizations were performed by the two-tailed Mann-Whitney test. (D to F) Vaginal gC2 IgG, gD2 IgG, or gE2 IgG ELISA titers: n = 10 for PolyC RNA and gD2 mRNA, n = 25 for trivalent mRNA, n = 10 for trivalent subunit protein. OD, optical density. (G) HSV-2 strain MS neutralizing antibody titers in the presence of 10% HSV-1/HSV-2 seronegative human serum as a source of complement. (H) HSV-1 strain NS neutralizing antibody titers in the presence of 10% seronegative human serum as a source of complement. n = 10 in the PolyC RNA group, n = 10 in the gD2 mRNA group, n = 20 in the trivalent mRNA group, and n = 10 in the trivalent subunit group in (G) and (H). (D to H) P values comparing trivalent mRNA with trivalent subunit protein or with gD2 mRNA were calculated by the two-tailed Mann-Whitney test. (I) ADCC using sera from PolyC RNA, gD2 mRNA, trivalent mRNA, and trivalent subunit mice. Results shown represent five to six separate sera tested per group. P < 0.001, comparing each curve with PolyC RNA as calculated by two-way ANOVA with Tukey for significance.

  • Fig. 5 Antibodies that block gC2 and gE2 immune evasion domains and that bind gD2 epitopes that mediate crucial functions.

    P values are shown in the figures for the primary end point comparison between trivalent mRNA and trivalent protein. Other P values are noted below. (A) Antibodies produced by trivalent mRNA and trivalent subunit protein block C3b binding to gC2. Error bars represent geometric means and 95% confidence intervals. P = 0.0220 (at 10 μg/ml) and P < 0.0001 (at 20 and 50 μg/ml), comparing trivalent mRNA with gD2 mRNA. P < 0.0001 (at 50 μg/ml) and P = not significant (at other IgG concentrations), comparing trivalent subunit protein with gD2 mRNA. (B) Blocking gE2 binding to IgG Fc. P = 0.0079, comparing trivalent mRNA with gD2 mRNA at 4 μg/ml. P = 0.1508 (at 4 μg/ml) and P = 0.0079 (at 2 μg/ml), comparing trivalent subunit protein with gD2 mRNA. (A and B) Results are geometric means with 95% CI. IgG was purified from pooled sera of 10 animals per group. Each entry represents results from a single assay. P values were determined by the two-tailed Mann-Whitney test. (C to H) Sera were evaluated for blocking gD2 binding to prototype mAbs that recognize crucial gD2 epitopes; n = 10 for PolyC RNA, n = 20 for trivalent mRNA, and n = 9 for trivalent subunit protein. Results in (C) to (G) represent means with 95% CI. P values comparing trivalent mRNA with trivalent subunit protein were calculated using the two-tailed Mann-Whitney test.

  • Fig. 6 CD4+, CD8+, TFH+ T cell responses and GC B cell responses.

    Splenocytes for CD4+ and CD8+ T cell assays were pooled from five animals, and the responses of six replicate wells are shown. (A) CD4+ T cell IFNγ responses in mice immunized with trivalent mRNA or (B) CD4+ polyfunctional IFNγ and TNFα responses in mice immunized with trivalent mRNA. (C) CD8+ T cell IFNγ responses in mice immunized with trivalent mRNA. (D) CD4+ T cell IFNγ responses in mice immunized with trivalent subunit protein or (E) CD4+ polyfunctional IFNγ and TNFα responses in mice immunized with trivalent subunit protein. (F) CD8+ T cell IFNγ responses in mice immunized with trivalent subunit protein. (A to F) Each peptide-stimulated CD4+ or CD8+ T cell response was compared with DMSO vehicle-treated controls. The CD4+ and CD8+ T cell assays for trivalent mRNA and trivalent subunit protein were run on separate days, which accounts for the variability in the DMSO control results. The percentage of cytokine positive cells is based on the total number of CD4+ T cells counted in the trivalent mRNA and subunit protein assays. The mean numbers of CD4+ T cells counted in the trivalent mRNA experiments and in the trivalent subunit protein were 4680 ± 714 and 7850 ± 960, respectively. The mean numbers of CD8+ T cells counted in the trivalent mRNA experiments and in the trivalent subunit protein were 981 ± 232 and 1118 ± 206, respectively. All values with P ≤ 0.05 are shown in the figures. P values were calculated by the Kruskal-Wallis test with Dunn’s for multiple comparisons. (G and H) TFH and GC B cell responses that include CpG/alum as a control. Splenocytes were harvested for TFH and GC B cell assays from individual mice 10 days after a single immunization with CpG/alum alone, trivalent mRNA-LNP, trivalent subunit protein–CpG/alum, or no immunization. P values are shown in the figures for the primary end point comparison between trivalent mRNA and trivalent protein. Other P values are noted below. (G) TFH cells: P = 0.532, comparing CpG/alum with trivalent subunit protein–CpG/alum. (H) GC B cells: P = 0.90, comparing CpG/alum with trivalent subunit protein. n = 5 animals per group, except n = 4 for CpG/alum. P values were calculated using the two-tailed Mann-Whitney test. Error bars represent geometric means and 95% confidence intervals. (I and J) TFH and GC B cell responses that include PolyC RNA-LNP as a control. Splenocytes were harvested for TFH and GC B cell assays from individual mice 10 days after the second immunization with PolyC RNA-LNP, trivalent mRNA-LNP, or no immunization. P values are shown in the figures and were calculated by the two-tailed Mann-Whitney test; n = 5 animals per group.

  • Table 1 Combined ID and IM efficacy studies.

    OutcomeNumber of animals with outcome*
    PolyC RNAgD2 mRNATrivalent mRNATrivalent protein
    Clinical disease:
    Death, genital lesions, or
    generalized illness
    25/251/100/440/20
    P = 0.1852P = 1.0000
    Subclinical infection
      Day 2 vaginal titers25/254/200/644/20
    P = 0.0025P = 0.0025
      Day 4 vaginal titers25/253/200/644/20
    P = 0.0120P = 0.0025
      HSV-2 DNA in DRG19/193/101/395/20
    P = 0.0231P = 0.0143
    Sterile immunity:
    No clinical or subclinical
    infection
    0/2515/2063/6423/30
    P = 0.0025P = 0.0012

    *The primary end point is the comparison between trivalent mRNA-LNP and trivalent subunit protein. The secondary end point is the comparison between trivalent mRNA-LNP and gD2 mRNA-LNP. P values were calculated by two-tailed Fisher’s exact test.

    Supplementary Materials

    • immunology.sciencemag.org/cgi/content/full/4/39/eaaw7083/DC1

      Fig. S1. Western blot and ELISA demonstrating expression and secretion of gC2, gD2, and gE2 proteins after transfection with nucleoside-modified mRNA-LNP.

      Fig. S2. Transmission studies.

      Fig. S3. IgG1 and IgG2a titers.

      Fig. S4. Flow cytometry gating strategy for the TFH and GC B cells.

      Data file S1. Raw data (Excel).

      References (5559)

    • Supplementary Materials

      The PDF file includes:

      • Fig. S1. Western blot and ELISA demonstrating expression and secretion of gC2, gD2, and gE2 proteins after transfection with nucleoside-modified mRNA-LNP.
      • Fig. S2. Transmission studies.
      • Fig. S3. IgG1 and IgG2a titers.
      • Fig. S4. Flow cytometry gating strategy for the TFH and GC B cells.

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      Other Supplementary Material for this manuscript includes the following:

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