Yujuan Yue

A vaccine based on the rhesus cytomegalovirus UL128 complex induces broadly neutralizing antibodies in rhesus macaques

ABSTRACT Neutralizing antibodies (NAb) are important for interfering with horizontal transmission of human cytomegalovirus (HCMV) leading to primary and congenital HCMV infection. Recent findings have shown that a pentameric virion complex formed by the glycoproteins gH/gL, UL128, UL130, and UL131A (UL128C) is required for HCMV entry into epithelial/endothelial cells (Epi/EC) and is the target of potent NAb in HCMV-seropositive individuals. Using bacterial artificial chromosome technology, we have generated a modified vaccinia Ankara virus (MVA) that stably coexpresses all 5 rhesus CMV (RhCMV) proteins homologous to HCMV UL128C, termed MVA-RhUL128C. Coimmunoprecipitation confirmed the interaction of RhgH with the other 4 RhCMV subunits of the pentameric complex. All 8 RhCMV-naïve rhesus macaques (RM) vaccinated with MVA-RhUL128C developed NAb that blocked infection of monkey kidney epithelial cells (MKE) and rhesus fibroblasts. NAb titers induced by MVA-RhUL128C measured on both cell types at 2 to 6 weeks postvaccination were comparable to levels observed in naturally infected RM. In contrast, MVA expressing a subset of RhUL128C proteins or RhgB glycoprotein only minimally stimulated NAb that inhibited infection of MKE. In addition, following subcutaneous RhCMV challenge at 8 weeks postvaccination, animals vaccinated with MVA-RhUL128C showed reduced plasma viral loads. These results indicate that MVA expressing the RhUL128C induces NAb inhibiting RhCMV entry into both Epi/EC and fibroblasts and limits RhCMV replication in RM. This novel approach is the first step in developing a prophylactic HCMV vaccine designed to interfere with virus entry into major cell types permissive for viral replication, a required property of an effective vaccine.

Chapter 5 Rhesus Cytomegalovirus: A Nonhuman Primate Model for The Study of Human Cytomegalovirus

Human cytomegalovirus (HCMV), a member of an ancient family of viruses (Herpesviridae), has acquired the capacity to maintain a lifelong persistent infection within an immunocompetent host. Since both primary and recurrent infections are generally subclinical, host antiviral immune responses are effective at limiting the pathogenic potential of HCMV. However, the fact that HCMV can persist in the presence of those protective immune responses indicates that host immunity is unable to prevent or eliminate long-term reservoirs of virus. The ability of HCMV to persist has important clinical implications, a fact reflected by the spectrum of pathogenic outcomes observed in those without a fully functional immune system. Recurrence of viral replication or transmission of HCMV from an infected individual to those most susceptible to primary infection during immune suppression, deficiency, or immaturity can lead to multiorgan disease and, sometimes, death. The clinical need for a protective HCMV vaccine has been recognized for decades, but due to a conspiracy of factors, there is no approved vaccine despite intensive investigations to develop one. Animal models of HCMV have been used as systems of discovery and translation to understand viral mechanisms of persistence and pathogenesis, and to test concepts and modalities for the generation of immune responses that protect from primary infection and sequelae. This review summarizes studies in a nonhuman primate model of HCMV involving infection of rhesus macaques (Macaca mulatta) with rhesus cytomegalovirus (RhCMV). The RhCMV model serves as an important complement to those in other animals, particularly small animals, and the lessons learned from RhCMV should have direct clinical relevance to HCMV and the design of protective vaccines.

Immunogenicity and Protective Efficacy of DNA Vaccines Expressing Rhesus Cytomegalovirus Glycoprotein B, Phosphoprotein 65-2, and Viral Interleukin-10 in Rhesus Macaques

ABSTRACT Rhesus cytomegalovirus (RhCMV) infection of macaques exhibits strong similarities to human CMV (HCMV) persistence and pathogenesis. The immunogenicity of DNA vaccines encoding three RhCMV proteins (a truncated version of glycoprotein B lacking the transmembrane region and endodomain [gBΔTM], phosphoprotein 65-2 [pp65-2], and viral interleukin-10 [vIL-10]) was evaluated in rhesus macaques. Two groups of monkeys (four per group) were genetically immunized four times with a mixture of either pp65-2 and gBΔTM or pp65-2, vIL-10, and gBΔTM. The vaccinees developed anti-gB and anti-pp65-2 antibodies in addition to pp65-2 cellular responses after the second booster immunization, with rapid responses observed with subsequent DNA injections. Weak vIL-10 immune responses were detected in two of the four immunized animals. Neutralizing antibodies were detected in seven monkeys, although titers were weak compared to those observed in naturally infected animals. The immunized monkeys and naïve controls were challenged intravenously with 105 PFU of RhCMV. Anamnestic binding and neutralizing antibody responses were observed 1 week postchallenge in the vaccinees. DNA vaccination-induced immune responses significantly decreased peak viral loads in the immunized animals compared to those in the controls. No difference in peak viral loads was observed between the pp65-2/gBΔTM DNA- and pp65-2/vIL-10/gBΔTM-vaccinated groups. Antibody responses to nonvaccine antigens were lower postchallenge in both vaccine groups than in the controls, suggesting long-term control of RhCMV protein expression. These data demonstrated that DNA vaccines targeting the RhCMV homologues of HCMV gB and pp65 altered the course of acute and persistent RhCMV infection in a primate host.

Evaluation of recombinant modified vaccinia Ankara virus-based rhesus cytomegalovirus vaccines in rhesus macaques

A vaccine consisting of rhesus cytomegalovirus (RhCMV) pp65-2, gB and IE1 expressed via modified vaccinia Ankara (MVA) was evaluated in rhesus macaques with or without prior priming with expression plasmids for the same antigens. Following two MVA treatments, comparable levels of anti-gB, pp65-2 and neutralizing antibody responses, and pp65-2- and IE1-specific cellular immune responses were detected in both vaccinated groups. Similar reductions in plasma peak viral loads were observed in these groups compared to untreated controls. This study demonstrates the immunogenicity and protective efficacy of rMVA-based RhCMV subunit vaccines in a primate host and warrants further investigation to improve the efficacy of subunit vaccines against CMV.

Vaccine-Induced Control of Viral Shedding following Rhesus Cytomegalovirus Challenge in Rhesus Macaques

ABSTRACT The use of animal models of human cytomegalovirus (HCMV) infection is critical to refine HCMV vaccine candidates. Previous reports have demonstrated that immunization of rhesus monkeys against rhesus cytomegalovirus (RhCMV) can reduce both local and systemic replication of RhCMV following experimental RhCMV challenge. These studies used prime/boost combinations of DNA expression plasmids alone or DNA priming and boosting with either inactivated virion particles or modified vaccinia virus Ankara (MVA) expressing the same antigens. Viral outcomes included reduced RhCMV replication at the site of subcutaneous inoculation and RhCMV viremia following intravenous inoculation. Since shedding of cytomegalovirus from mucosal surfaces is critical for horizontal transmission of the virus, DNA priming/MVA boosting was evaluated for the ability to reduce oral shedding of RhCMV following subcutaneous challenge. Of six rhesus monkeys vaccinated exclusively against RhCMV glycoprotein B (gB), phosphoprotein 65 (pp65), and immediate-early 1 (IE1), half showed viral loads in saliva that were lower than those of control monkeys by 1 to 3 orders of magnitude. Further, there was a strong association of memory pp65 T cell responses postchallenge in animals exhibiting the greatest reduction in oral shedding. These results highlight the fact that a DNA/MVA vaccination regimen can achieve a notable reduction in a critical parameter of viral replication postchallenge. The recently completed clinical trial of a gB subunit vaccine in which the rate of HCMV infection was reduced by 50% in the individuals receiving the vaccine is consistent with the results of this study suggesting that additional immunogens are likely essential for maximum protection in an outbred human population.

Open Reading Frames Carried on UL/b′ Are Implicated in Shedding and Horizontal Transmission of Rhesus Cytomegalovirus in Rhesus Monkeys

ABSTRACT Implicit with the use of animal models to test human cytomegalovirus (HCMV) vaccines is the assumption that the viral challenge of vaccinated animals reflects the anticipated virus-host interactions following exposure of vaccinated humans to HCMV. Variables of animal vaccine studies include the route of exposure to and the titer of challenge virus, as well as the genomic coding content of the challenge virus. This study was initiated to provide a better context for conducting vaccine trials with nonhuman primates by determining whether the in vivo phenotype of culture-passaged strains of rhesus cytomegalovirus (RhCMV) is comparable to that of wild-type RhCMV (RhCMV-WT), particularly in relation to the shedding of virus into bodily fluids and the potential for horizontal transmission. Results of this study demonstrate that two strains containing a full-length UL/b′ region of the RhCMV genome, which encodes proteins involved in epithelial tropism and immune evasion, were persistently shed in large amounts in bodily fluids and horizontally transmitted, whereas a strain lacking a complete UL/b′ region was not shed or transmitted to cagemates. Shedding patterns exhibited by strains encoding a complete UL/b′ region were consistent with patterns observed in naturally infected monkeys, the majority of whom persistently shed high levels of virus in saliva for extended periods of time after seroconversion. Frequent viral shedding contributed to a high rate of infection, with RhCMV-infected monkeys transmitting virus to one naïve animal every 7 weeks after introduction of RhCMV-WT into an uninfected cohort. These results demonstrate that the RhCMV model can be designed to rigorously reflect the challenges facing HCMV vaccine trials, particularly those related to horizontal transmission.

Maternal CD4+ T cells protect against severe congenital cytomegalovirus disease in a novel nonhuman primate model of placental cytomegalovirus transmission

Significance Congenital cytomegalovirus (CMV) is the leading infectious cause of childhood hearing loss and brain damage worldwide. Yet, despite its high prevalence and ranking as a top priority for vaccine development, the immune correlates of protection that could guide vaccine development remain undefined. Using a novel nonhuman primate model of congenital CMV transmission, we demonstrate a critical role for maternal CD4+ T cells in the induction of protective maternal immune responses that prevent fetal demise. In addition to establishing placental CMV transmission for the first time (to our knowledge) in nonhuman primates, this study reveals an association between delayed maternal virus-specific neutralizing antibody responses and severe fetal outcome, providing insight into the mechanism by which maternal CD4+ T cells impact congenital CMV disease. Elucidation of maternal immune correlates of protection against congenital cytomegalovirus (CMV) is necessary to inform future vaccine design. Here, we present a novel rhesus macaque model of placental rhesus CMV (rhCMV) transmission and use it to dissect determinants of protection against congenital transmission following primary maternal rhCMV infection. In this model, asymptomatic intrauterine infection was observed following i.v. rhCMV inoculation during the early second trimester in two of three rhCMV-seronegative pregnant females. In contrast, fetal loss or infant CMV-associated sequelae occurred in four rhCMV-seronegative pregnant macaques that were CD4+ T-cell depleted at the time of inoculation. Animals that received the CD4+ T-cell–depleting antibody also exhibited higher plasma and amniotic fluid viral loads, dampened virus-specific CD8+ T-cell responses, and delayed production of autologous neutralizing antibodies compared with immunocompetent monkeys. Thus, maternal CD4+ T-cell immunity during primary rhCMV infection is important for controlling maternal viremia and inducing protective immune responses that prevent severe CMV-associated fetal disease.

A heterologous DNA prime/protein boost immunization strategy for rhesus cytomegalovirus

A previous study in nonhuman primates demonstrated that genetic immunization against the rhesus cytomegalovirus phosphoprotein 65-2 (pp65-2) and glycoprotein B (gB) antigens both stimulated antigen-specific antibodies and CD8 T cell responses, and significantly reduced plasma viral loads following intravenous challenge with RhCMV. It was also noted in this study that weak CD4 T cell and neutralizing antibody responses were generated by DNA alone. To broaden the type of immune responses, a DNA prime/protein boost strategy was used in seronegative macaques, consisting of four DNA immunizations against pp65-2, gB, and immediate-early 1 (IE1), followed by two boosts with formalin-inactivated RhCMV virions. This heterologous prime/boost strategy elicited robust antigen-specific CD4 and CD8 T cell responses in addition to biologically relevant neutralizing antibody titers. Animals were challenged with RhCMV delivered into four sites via a subcutaneous route. Skin biopsies of one of the inoculation sites 7 days post challenge revealed marked differences in the level of RhCMV replication between the vaccinated and control monkeys. Whereas the inoculation site in the controls was noted for a prominent inflammatory response and numerous cytomegalic, antigen-positive (IE1) cells, the inoculation site in the vaccinees was characterized by an absence of inflammation and antigen-positive cells. All five vaccinees developed robust recall responses to viral antigens, and four of them exhibited long-term viral immune responses consistent with effective control of viral expression and replication. These results demonstrate that a heterologous DNA prime/protein boost strategy greatly expands the breadth of antiviral immune responses and greatly reduces the level of viral replication at the primary site of challenge infection.

Host Immune Responses to a Viral Immune Modulating Protein: Immunogenicity of Viral Interleukin-10 in Rhesus Cytomegalovirus-Infected Rhesus Macaques

Background Considerable evidence has accumulated that multiple viruses, bacteria, and protozoa manipulate interleukin-10 (IL-10)-mediated signaling through the IL-10 receptor (IL-10R) in ways that could enable establishment of a persistent microbial infection. This suggests that inhibition of pathogen targeting of IL-10/IL-10R signaling could prevent microbial persistence. Human cytomegalovirus (HCMV) and rhesus cytomegalovirus (RhCMV) express a viral interleukin-10 (cmvIL-10 and rhcmvIL-10, respectively) with comparable immune modulating properties in vitro to that of their hosts cellular IL-10 (cIL-10). A prior study noted that rhcmvIL-10 alters innate and adaptive immunity to RhCMV in vivo, consistent with a central role for rhcmvIL-10 during acute virus-host interactions. Since cmvIL-10 and rhcmvIL-10 are extremely divergent from the cIL-10 of their respective hosts, vaccine-mediated neutralization of their function could inhibit establishment of viral persistence without inhibition of cIL-10. Methods and Findings As a prelude to evaluating cmvIL-10-based vaccines in humans, the rhesus macaque model of HCMV was used to interrogate peripheral and mucosal immune responses to rhcmvIL-10 in RhCMV-infected animals. ELISA were used to detect rhcmvIL-10-binding antibodies in plasma and saliva, and an IL-12-based bioassay was used to quantify plasma antibodies that neutralized rhcmvIL-10 function. rhcmvIL-10 is highly immunogenic during RhCMV infection, stimulating high avidity rhcmvIL-10-binding antibodies in the plasma of all infected animals. Most infected animals also exhibited plasma antibodies that partially neutralized rhcmvIL-10 function but did not cross-neutralize the function of rhesus cIL-10. Notably, minimally detectable rhcmvIL-10-binding antibodies were detected in saliva. Conclusion This study demonstrates that rhcmvIL-10, as a surrogate for cmvIL-10, is a viable vaccine candidate because (1) it is highly immunogenic during natural RhCMV infection, and (2) neutralizing antibodies to rhcmvIL-10 do not cross-react with rhesus cIL-10. Exceedingly low rhcmvIL-10 antibodies in saliva further suggest that the oral mucosa, which is critical in RhCMV natural history, is associated with suboptimal anti-rhcmvIL-10 antibody responses.