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Clinical Trial
. 2019 Nov 13;93(23):e00747-19.
doi: 10.1128/JVI.00747-19. Print 2019 Dec 1.

A Replication-Defective Human Cytomegalovirus Vaccine Elicits Humoral Immune Responses Analogous to Those with Natural Infection

Yaping Liu #  1 , Daniel C Freed #  1 , Leike Li #  2 , Aimin Tang  1 , Fengsheng Li  1 , Edward M Murray  1 , Stuart P Adler  3   4 , Michael A McVoy  4 , Richard E Rupp  5 , Diane Barrett  5 , Xiaohua Ye  2 , Ningyan Zhang  2 , Karen Beck  1 , Timothy Culp  1 , Rituparna Das  1 , Liping Song  1 , Kalpit Vora  1 , Hua Zhu  6 , Dai Wang  7 , Amy S Espeseth  1 , Zhiqiang An  2 , Luwy Musey  1 , Tong-Ming Fu  7   2
Affiliations
Clinical Trial

A Replication-Defective Human Cytomegalovirus Vaccine Elicits Humoral Immune Responses Analogous to Those with Natural Infection

Yaping Liu et al. J Virol. .

Abstract

Human cytomegalovirus (HCMV) can cause congenital infections, which are a leading cause of childhood disabilities. Since the rate of maternal-fetal transmission is much lower in naturally infected (HCMV-seropositive) women, we hypothesize that a vaccine candidate capable of eliciting immune responses analogous to those of HCMV-seropositive subjects may confer protection against congenital HCMV. We have previously described a replication-defective virus vaccine based on strain AD169 (D. Wang, D. C. Freed, X. He, F. Li, et al., Sci Transl Med 8:362ra145, 2016, https://doi.org/10.1126/scitranslmed.aaf9387). The vaccine, named V160, has been shown to be safe and immunogenic in HCMV-seronegative human subjects, eliciting both humoral and cellular immune responses (S. P. Adler, S. E. Starr, S. A. Plotkin, S. H. Hempfling, et al., J Infect Dis 220:411-419, 2019, https://doi.org/10.1093/infdis/171.1.26). Here, we further showed that sera from V160-immunized HCMV-seronegative subjects have attributes similar in quality to those from seropositive subjects, including high-avidity antibodies to viral antigens, coverage against a panel of genetically distinct clinical isolates, and protection against viral infection in diverse types of human cells in culture. More importantly, vaccination appeared efficient in priming the human immune system, inducing memory B cells in six V160 recipients at frequencies comparable to those of three HCMV-seropositive subjects. Our results demonstrate the ability of V160 to induce robust and durable humoral memory responses to HCMV, justifying further clinical evaluation of the vaccine against congenital HCMV.IMPORTANCEIn utero HCMV infection can lead to miscarriage or childhood disabilities, and an effective vaccine is urgently needed. Since children born to women who are seropositive prior to pregnancy are less likely to be affected by congenital HCMV infection, it has been hypothesized that a vaccine capable of inducing an immune response resembling the responses in HCMV-seropositive women may be effective. We previously described a replication-defective virus vaccine that has been demonstrated safe and immunogenic in HCMV-seronegative subjects. Here, we conducted additional analyses to show that the vaccine can induce antibodies with functional attributes similar to those from HCMV-seropositive subjects. Importantly, vaccination can induce long-lived memory B cells at frequencies comparable to those seen in HCMV-seropositive subjects. We conclude that this vaccine is a promising candidate that warrants further clinical evaluation for prevention of congenital HCMV.

Keywords: CMV; V160; humoral immunity; memory; neutralization; vaccine.

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Figures

FIG 1
FIG 1
Longitudinal neutralizing titers of V160-vaccinated groups in ARPE-19 and MRC-5 cells. Serological neutralizing titers for vaccine groups were assessed in ARPE-19 (A) or MRC-5 cells (B). The reciprocal serum dilution that can achieve 50% viral neutralization (NT50) was calculated using four-parameter curve fitting, and the geometric mean titers (GMTs) for each group are plotted along with standard error (se). The horizontal line represents the GMTs determined from 53 HCMV-seropositive subjects.
FIG 2
FIG 2
Schematic drawing for culturing memory B cells to assess their HCMV specificity.
FIG 3
FIG 3
Frequency of HCMV-specific memory B cells in seropositive and vaccine subjects. PBMC samples were cultured for antibody production as outlined in Fig. 2. (A) Each quadrant graph represents the results for a single subject, and each dot represents data for a clonal B-cell culture supernatant that was assessed for binding to HCMV in ELISA (y axis) and the ability to neutralize virus (x axis). Data for three HCMV-seropositive subjects are shown in blue. Six vaccine subjects received 30 U of V160 either i.m. (yellow) or i.d. (red) at months 0, 1, and 6. PBMC samples were collected at month 12 for this analysis. Positive/negative cutoffs were set at 10-fold above background for binding ELISA and at 50% for neutralization. The adjusted HCMV-specific B-cell frequencies normalized by the IgG production rate of each subject (see Materials and Methods) are marked in each quadrant. (B) Predicted mean values and 95% confidence intervals of adjusted B-cell frequencies for the three donor groups in ELISA and neutralization assays, as indicated. Statistical analyses were conducted using a mixed-effect logistic regression.
FIG 4
FIG 4
Broadly neutralizing antibodies induced by V160 vaccination. Serum samples from the 100U+alum group were assessed in ARPE-19 (A) or MRC-5 (B) cells for their ability to neutralize a panel of 12 genetically characterized clinical isolates, along with Towne- and AD169-derived epithelial tropic laboratory strains. Serum samples from the six subjects who received 30 U of V160 either i.m. or i.d. as described in the legend of Fig. 3 were also assessed for neutralizing activities in ARPE-19 (C) or MRC-5 (D) cells. All vaccine immune sera are from study month 12.
FIG 5
FIG 5
Neutralizing activity of vaccine immune sera in diverse human cell types. Serum neutralizing activities were assessed in human oral mucosal epithelial cells (A), human umbilical vascular endothelial cells (HUVEC) (B), human cytotrophoblast cells (C), or human neuronal cells (D). Serum samples include month 12 sera from the 100U+alum group and six vaccine subjects who received 30 U either i.m. or i.d. as described in the legend of Fig. 3. Sera from two HCMV-seropositive donors and thee HCMV-seronegative donors were included as controls. Each dot represents a single serum sample, and each red line the GMT for the group. Comparison between groups was conducted using one-way ANOVA. n.s., not significant.
FIG 6
FIG 6
Vaccine-induced antibodies reactive to HCMV antigens. Serum endpoint dilution titers were determined using purified HCMV virions (CMV), recombinant gB, or recombinant pentameric complex as ELISA antigens. Longitudinal immune sera from 100U and 100U+alum groups were included along with sera from 10 seropositive donors and 10 seronegative donors. The GMTs for each group were plotted longitudinally. s.e., standard error.
FIG 7
FIG 7
High-avidity antibodies induced by V160 vaccination. Serum samples from the 100U and 100U+alum groups were evaluated longitudinally for their relative avidity indices to purified HCMV virions (CMV), recombinant gB, or pentameric complex. Sera were tested by ELISA at a 1:400 dilution, with or without urea. Avidity indices are presented longitudinally. The squares indicate the mean avidity indices of sera from HCMV-seropositive (+) subjects (n = 10), which are included as controls.
FIG 8
FIG 8
Inhibition of viral spread by V160 vaccine-induced immune sera. (A) HFF cells infected with AD169rev-GFP at a low MOI were treated with serum from subject 300194 at baseline (month 0) or postvaccination (month 12), and representative images from days 4 to 7 postinfection are shown. ARPE-19 cells (B) or HFF cells (C) were infected with HCMV as described above for 1 day and then mock treated or treated with 10 μM cidofovir (CDV) or treated with 1:40 dilutions of human sera from HCMV-seropositive subjects (n = 8), or with prevaccination (month 0) or postvaccination (month 12) sera from subjects vaccinated with either V160 in the regimen using 100 U or 100 U plus alum. Ro values were determined as described in Materials and Methods. Comparisons between groups were conducted using one-way ANOVA. n.s., not statistically significant.
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