Relationship of maternal cytomegalovirus-specific antibody responses and viral load to vertical transmission risk following primary maternal infection in a rhesus macaque model.

Cytomegalovirus (CMV) is the most common congenital infection and cause of birth defects worldwide. Primary CMV infection during pregnancy leads to a higher frequency of congenital CMV (cCMV) than maternal re-infection, suggesting that maternal immunity confers partial protection. However, poorly understood immune correlates of protection against placental transmission contributes to the current lack of an approved vaccine to prevent cCMV. In this study, we characterized the kinetics of maternal plasma rhesus CMV (RhCMV) viral load (VL) and RhCMV-specific antibody binding and functional responses in a group of 12 immunocompetent dams with acute, primary RhCMV infection. We defined cCMV transmission as RhCMV detection in amniotic fluid (AF) by qPCR. We then leveraged a large group of past and current primary RhCMV infection studies in late-first/early-second trimester RhCMV-seronegative rhesus macaque dams, including immunocompetent (n = 15), CD4+ T cell-depleted with (n = 6) and without (n = 6) RhCMV-specific polyclonal IgG infusion before infection to evaluate differences between RhCMV AF-positive and AF-negative dams. During the first 3 weeks after infection, the magnitude of RhCMV VL in maternal plasma was higher in AF-positive dams in the combined cohort, while RhCMV glycoprotein B (gB)- and pentamer-specific binding IgG responses were lower magnitude compared to AF-negative dams. However, these observed differences were driven by the CD4+ T cell-depleted dams, as there were no differences in plasma VL or antibody responses between immunocompetent AF-positive vs AF-negative dams. Overall, these results suggest that levels of neither maternal plasma viremia nor humoral responses are associated with cCMV following primary maternal infection in healthy individuals. We speculate that other factors related to innate immunity are more important in this context as antibody responses to acute infection likely develop too late to influence vertical transmission. Yet, pre-existing CMV glycoprotein-specific and neutralizing IgG may provide protection against cCMV following primary maternal CMV infection even in high-risk, immunocompromised settings.

Pathogenesis of Wild-Type-Like Rhesus Cytomegalovirus Strains following Oral Exposure of Immune-Competent Rhesus Macaques.

Rhesus cytomegalovirus (RhCMV) infection of rhesus macaques (Macaca mulatta) is a valuable nonhuman primate model of human CMV (HCMV) persistence and pathogenesis. In vivo studies predominantly use tissue culture-adapted variants of RhCMV that contain multiple genetic mutations compared to wild-type (WT) RhCMV. In many studies, animals have been inoculated by nonnatural routes (e.g., subcutaneous, intravenous) that do not recapitulate disease progression via the normative route of mucosal exposure. Accordingly, the natural history of RhCMV would be more accurately reproduced by infecting macaques with strains of RhCMV that reflect the WT genome using natural routes of mucosal transmission. Here, we tested two WT-like RhCMV strains, UCD52 and UCD59, and demonstrated that systemic infection and frequent, high-titer viral shedding in bodily fluids occurred following oral inoculation. RhCMV disseminated to a broad range of tissues, including the central nervous system and reproductive organs. Commonly infected tissues included the thymus, spleen, lymph nodes, kidneys, bladder, and salivary glands. Histological examination revealed prominent nodular hyperplasia in spleens and variable levels of lymphoid lymphofollicular hyperplasia in lymph nodes. One of six inoculated animals had limited viral dissemination and shedding, with commensurately weak antibody responses to RhCMV antigens. These data suggest that long-term RhCMV infection parameters might be restricted by local innate factors and/or de novo host immune responses in a minority of primary infections. Together, we have established an oral RhCMV infection model that mimics natural HCMV infection. The virological and immunological parameters characterized in this study will greatly inform HCMV vaccine designs for human immunization. IMPORTANCE Human cytomegalovirus (HCMV) is globally ubiquitous with high seroprevalence rates in all communities. HCMV infections can occur vertically following mother-to-fetus transmission across the placenta and horizontally following shedding of virus in bodily fluids in HCMV-infected hosts and subsequent exposure of susceptible individuals to virus-laden fluids. Intrauterine HCMV has long been recognized as an infectious threat to fetal growth and development. Since vertical HCMV infections occur following horizontal HCMV transmission to the pregnant mother, the nonhuman primate model of HCMV pathogenesis was used to characterize the virological and immunological parameters of infection following primary mucosal exposures to rhesus cytomegalovirus.

Horizontal Transmission of Cytomegalovirus in a Rhesus Model Despite High-Level, Vaccine-Elicited Neutralizing Antibody and T-Cell Responses.

The development of a vaccine to prevent congenital human cytomegalovirus (HCMV) disease is a public health priority. We tested rhesus CMV (RhCMV) prototypes of HCMV vaccine candidates in a seronegative macaque oral challenge model. Immunogens included a recombinant pentameric complex (PC; gH/gL/pUL128/pUL130/pUL131A), a postfusion gB ectodomain, and a DNA plasmid that encodes pp65-2. Immunization with QS21-adjuvanted PC alone or with the other immunogens elicited neutralizing titers comparable to those elicited by RhCMV infection. Similarly, immunization with all 3 immunogens elicited pp65-specific cytotoxic T-cell responses comparable to those elicited by RhCMV infection. RhCMV readily infected immunized animals and was detected in saliva, blood, and urine after challenge in quantities similar to those in placebo-immunized animals. If HCMV evades vaccine-elicited immunity in humans as RhCMV evaded immunity in macaques, a HCMV vaccine must elicit immunity superior to, or different from, that elicited by the prototype RhCMV vaccine to block horizontal transmission.

In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome.

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68-1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68-1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques.

Neutralization of rhesus cytomegalovirus IL-10 reduces horizontal transmission and alters long-term immunity.

Human cytomegalovirus (HCMV) causes severe disease in infants and immunocompromised people. There is no approved HCMV vaccine, and vaccine development strategies are complicated by evidence of both persistent infection and reinfection of people with prior immunity. The greatest emphasis has been placed on reducing transmission to seronegative pregnant women to prevent vertical transmission and its potentially severe sequelae. Increasing evidence suggests that the earliest host-HCMV interactions establish conditions for viral persistence, including evasion of host immune responses to the virus. Using a nonhuman primate model of HCMV infection, we show that rhesus macaques immunized against viral interleukin-10 (IL-10) manifest delayed rhesus cytomegalovirus (RhCMV) acquisition and altered immune responses to the infection when it does occur. Among animals with the greatest antiviral IL-10-neutralizing activity, the timing of RhCMV seroconversion was delayed by an average of 12 weeks. After acquisition, such animals displayed an antibody response to the new infection, which peaked as expected after 2 weeks but then declined rapidly. In contrast, surprisingly, vaccination with glycoprotein B (gB) protein had no discernible impact on these outcomes. Our results demonstrate that viral IL-10 is a key regulator of successful host immune responses to RhCMV. Viral IL-10 is, therefore, an important target for vaccine strategies against cytomegalovirus (CMV). Furthermore, given the immunoregulatory function of viral IL-10, targeting this protein may prove synergistic with other vaccine therapies and targets. Our study also provides additional evidence that the earliest host-CMV interactions can have a significant impact on the nature of persistent infection.

Exploitation of Interleukin-10 (IL-10) Signaling Pathways: Alternate Roles of Viral and Cellular IL-10 in Rhesus Cytomegalovirus Infection.

There is accumulating evidence that the viral interleukin-10 (vIL-10) ortholog of both human and rhesus cytomegalovirus (HCMV and RhCMV, respectively) suppresses the functionality of cell types that are critical to contain virus dissemination and help shape long-term immunity during the earliest virus-host interactions. In particular, exposure of macrophages, peripheral blood mononuclear cells, monocyte-derived dendritic cells, and plasmacytoid dendritic cells to vIL-10 suppresses multiple effector functions including, notably, those that link innate and adaptive immune responses. Further, vaccination of RhCMV-uninfected rhesus macaques with nonfunctional forms of RhCMV vIL-10 greatly restricted parameters of RhCMV infection following RhCMV challenge of the vaccinees. Vaccinees exhibited significantly reduced shedding of RhCMV in saliva and urine following RhCMV challenge compared to shedding in unvaccinated controls. Based on the evidence that vIL-10 is critical during acute infection, the role of vIL-10 during persistent infection was analyzed in rhesus macaques infected long term with RhCMV to determine whether postinfection vaccination against vIL-10 could change the virus-host balance. RhCMV-seropositive macaques, which shed RhCMV in saliva, were vaccinated with nonfunctional RhCMV vIL-10, and shedding levels of RhCMV in saliva were evaluated. Following robust increases in vIL-10-binding and vIL-10-neutralizing antibodies, shedding levels of RhCMV modestly declined, consistent with the interpretation that vIL-10 may play a functional role during persistent infection. However, a more significant association was observed between the levels of cellular IL-10 secreted in peripheral blood mononuclear cells exposed to RhCMV antigens and shedding of RhCMV in saliva. This result implies that RhCMV persistence is associated with the induction of cellular IL-10 receptor-mediated signaling pathways. IMPORTANCE: Human health is adversely impacted by viruses that establish lifelong infections that are often accompanied with increased morbidity and mortality (e.g., infections with HIV, hepatitis C virus, or human cytomegalovirus). A longstanding but unfulfilled goal has been to develop postinfection vaccine strategies that could "reboot" the immune system of an infected individual in ways that would enable the infected host to develop immune responses that clear reservoirs of persistent virus infection, effectively curing the host of infection. This concept was evaluated in rhesus macaques infected long term with rhesus cytomegalovirus by repeatedly immunizing infected animals with nonfunctional versions of the rhesus cytomegalovirus-encoded viral interleukin-10 immune-modulating protein. Following vaccine-mediated boosting of antibody titers to viral interleukin-10, there was modest evidence for increased immunological control of the virus following vaccination. More significantly, data were also obtained that indicated that rhesus cytomegalovirus is able to persist due to upregulation of the cellular interleukin-10 signaling pathway.

Sequencing the transcriptome of milk production: milk trumps mammary tissue.

BACKGROUND: Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, this assay has not been adequately validated in primates. Thus, the objectives of the current study were to assess the suitability of lactating rhesus macaques as a model for lactating humans and to determine whether RNA extracted from milk fractions is representative of RNA extracted from mammary tissue for the purpose of studying the transcriptome of milk-producing cells. RESULTS: We confirmed that macaque milk contains cytoplasmic crescents and that ample high-quality RNA can be obtained for sequencing. Using RNA sequencing, RNA extracted from macaque milk fat and milk cell fractions more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from whole mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat, whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from milk-producing mammary epithelial cells. CONCLUSIONS: RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells in a non-human primate. However, this sample type clearly requires protocols that minimize RNA degradation. Overall, we validated the use of RNA extracted from human and macaque milk and provided evidence to support the use of lactating macaques as a model for human lactation.

The pentameric complex is not required for vertical transmission of cytomegalovirus in seronegative pregnant rhesus macaques.

Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neonatal neurological impairment but essential virological determinants of transplacental CMV transmission remain unclear. The pentameric complex (PC), composed of five subunits, glycoproteins H (gH), gL, UL128, UL130, and UL131A, is essential for efficient entry into non-fibroblast cells in vitro . Based on this role in cell tropism, the PC is considered a possible target for CMV vaccines and immunotherapies to prevent cCMV. To determine the role of the PC in transplacental CMV transmission in a non-human primate model of cCMV, we constructed a PC-deficient rhesus CMV (RhCMV) by deleting the homologues of the HCMV PC subunits UL128 and UL130 and compared congenital transmission to PC-intact RhCMV in CD4+ T cell-depleted or immunocompetent RhCMV-seronegative, pregnant rhesus macaques (RM). Surprisingly, we found that the transplacental transmission rate was similar for PC-intact and PC-deleted RhCMV based on viral genomic DNA detection in amniotic fluid. Moreover, PC-deleted and PC-intact RhCMV acute infection led to similar peak maternal plasma viremia. However, there was less viral shedding in maternal urine and saliva and less viral dissemination in fetal tissues in the PC-deleted group. As expected, dams inoculated with PC-deleted RhCMV demonstrated lower plasma IgG binding to PC-intact RhCMV virions and soluble PC, as well as reduced neutralization of PC-dependent entry of the PC-intact RhCMV isolate UCD52 into epithelial cells. In contrast, binding to gH expressed on the cell surface and neutralization of entry into fibroblasts by the PC-intact RhCMV was higher for dams infected with PC-deleted RhCMV compared to those infected with PC-intact RhCMV. Our data demonstrates that the PC is dispensable for transplacental CMV infection in our non-human primate model. One Sentence Summary: Congenital CMV transmission frequency in seronegative rhesus macaques is not affected by the deletion of the viral pentameric complex.

Relationship of maternal cytomegalovirus-specific antibody responses and viral load to vertical transmission risk following primary maternal infection in a rhesus macaque model.

Cytomegalovirus (CMV) is the most common congenital infection and cause of birth defects worldwide. Primary CMV infection during pregnancy leads to a higher frequency of congenital CMV (cCMV) than maternal re-infection, suggesting that maternal immunity confers partial protection. However, poorly understood immune correlates of protection against placental transmission contributes to the current lack of an approved vaccine to prevent cCMV. In this study, we characterized the kinetics of maternal plasma rhesus CMV (RhCMV) viral load (VL) and RhCMV-specific antibody binding and functional responses in a group of 12 immunocompetent dams with acute, primary RhCMV infection. We defined cCMV transmission as RhCMV detection in amniotic fluid (AF) by qPCR. We then leveraged a large group of past and current primary RhCMV infection studies in late-first/early-second trimester RhCMV-seronegative rhesus macaque dams, including immunocompetent (n=15), CD4+ T cell-depleted with (n=6) and without (n=6) RhCMV-specific polyclonal IgG infusion before infection to evaluate differences between RhCMV AF-positive and AF-negative dams. During the first 3 weeks after infection, the magnitude of RhCMV VL in maternal plasma was higher in AF-positive dams in the combined cohort, while RhCMV glycoprotein B (gB)- and pentamer-specific binding IgG responses were lower magnitude compared to AF-negative dams. However, these observed differences were driven by the CD4+ T cell-depleted dams, as there were no differences in plasma VL or antibody responses between immunocompetent AF-positive vs AF-negative dams. Overall, these results suggest that levels of neither maternal plasma viremia nor humoral responses are associated with cCMV following primary maternal infection in healthy individuals. We speculate that other factors related to innate immunity are more important in this context as antibody responses to acute infection likely develop too late to influence vertical transmission. Yet, pre-existing CMV glycoprotein-specific and neutralizing IgG may provide protection against cCMV following primary maternal CMV infection even in high-risk, immunocompromised settings.

Late gene expression-deficient cytomegalovirus vectors elicit conventional T cells that do not protect against SIV.

Rhesus cytomegalovirus-based (RhCMV-based) vaccine vectors induce immune responses that protect ~60% of rhesus macaques (RMs) from SIVmac239 challenge. This efficacy depends on induction of effector memory-based (EM-biased) CD8+ T cells recognizing SIV peptides presented by major histocompatibility complex-E (MHC-E) instead of MHC-Ia. The phenotype, durability, and efficacy of RhCMV/SIV-elicited cellular immune responses were maintained when vector spread was severely reduced by deleting the antihost intrinsic immunity factor phosphoprotein 71 (pp71). Here, we examined the impact of an even more stringent attenuation strategy on vector-induced immune protection against SIV. Fusion of the FK506-binding protein (FKBP) degradation domain to Rh108, the orthologue of the essential human CMV (HCMV) late gene transcription factor UL79, generated RhCMV/SIV vectors that conditionally replicate only when the FK506 analog Shield-1 is present. Despite lacking in vivo dissemination and reduced innate and B cell responses to vaccination, Rh108-deficient 68-1 RhCMV/SIV vectors elicited high-frequency, durable, EM-biased, SIV-specific T cell responses in RhCMV-seropositive RMs at doses of ≥ 1 × 106 PFU. Strikingly, elicited CD8+ T cells exclusively targeted MHC-Ia-restricted epitopes and failed to protect against SIVmac239 challenge. Thus, Rh108-dependent late gene expression is required for both induction of MHC-E-restricted T cells and protection against SIV.

Open reading frames carried on UL/b' are implicated in shedding and horizontal transmission of rhesus cytomegalovirus in rhesus monkeys.

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.

Vaccine-induced control of viral shedding following rhesus cytomegalovirus challenge in rhesus macaques.

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.

Cytomegalovirus-vectored vaccines for HIV and other pathogens.

: The use of cytomegalovirus (CMV) as a vaccine vector to express antigens against multiple infectious diseases, including simian immunodeficiency virus, Ebola virus, plasmodium, and mycobacterium tuberculosis, in rhesus macaques has generated extraordinary levels of protective immunity against subsequent pathogenic challenge. Moreover, the mechanisms of immune protection have altered paradigms about viral vector-mediated immunity against ectopically expressed vaccine antigens. Further optimization of CMV-vectored vaccines, particularly as this approach moves to human clinical trials will be augmented by a more complete understanding of how CMV engenders mechanisms of immune protection. This review summarizes the particulars of the specific CMV vaccine vector that has been used to date (rhesus CMV strain 68-1) in relation to CMV natural history.

Rapid dissemination of SIV following oral inoculation.

OBJECTIVE: To assess the earliest events regarding transmission and dissemination of SIV following nontraumatic oral inoculation in macaques. DESIGN: Juvenile and neonate rhesus macaques were orally inoculated with SIVmac251 and necropsied at 1, 2, 4, 7, or 14 days post-inoculation. Sites of transmission and the extent of viral spread were assessed by using molecular techniques and in situ hybridization to identify SIV nucleic acid in lymphoid and nonlymphoid tissues. RESULTS: This study demonstrates that 1 day post-exposure, SIV nucleic acid was detected in the alimentary canal only in tissues proximal to the stomach, including the oral and esophageal mucosa as well as the tonsils. Following infection, virus was observed to spread rapidly to regional and peripheral lymph nodes by 1 and 2 days post-inoculation (dpi). Hundreds of copies of SIV-DNA were detected 4 dpi, increasing to > 10 000 copies/1 x 10(6) cells by 7 dpi. Identification of SIV positive T cells and macrophages implicates these cell types in viral spread, although dissemination of free virus is also likely. CONCLUSIONS: Here the oral and esophageal mucosa, as well as tonsils, are demonstrated to be potential sites for viral infection upon nontraumatic oral exposure to SIV in macaques. The rapid dissemination following oral transmission observed in this study is reflective of SIV transmission across other mucosal surfaces. The rapidity with which SIV, and probably HIV, spreads throughout the lymphatics indicates a major obstacle for a vaccine amnestic immune response to eliminate infected cells prior to dissemination.

Immunogenicity of viral vector, prime-boost SIV vaccine regimens in infant rhesus macaques: attenuated vesicular stomatitis virus (VSV) and modified vaccinia Ankara (MVA) recombinant SIV vaccines compared to live-attenuated SIV.

In a previously developed infant macaque model mimicking HIV infection by breast-feeding, we demonstrated that intramuscular immunization with recombinant poxvirus vaccines expressing simian immunodeficiency virus (SIV) structural proteins provided partial protection against infection following oral inoculation with virulent SIV. In an attempt to further increase systemic but also local antiviral immune responses at the site of viral entry, we tested the immunogenicity of different orally administered, replicating vaccines. One group of newborn macaques received an oral prime immunization with a recombinant vesicular stomatitis virus expressing SIVmac239 gag, pol and env (VSV-SIVgpe), followed 2 weeks later by an intramuscular boost immunization with MVA-SIV. Another group received two immunizations with live-attenuated SIVmac1A11, administered each time both orally and intravenously. Control animals received mock immunizations or non-SIV VSV and MVA control vectors. Analysis of SIV-specific immune responses in blood and lymphoid tissues at 4 weeks of age demonstrated that both vaccine regimens induced systemic antibody responses and both systemic and local cell-mediated immune responses. The safety and immunogenicity of the VSV-SIVgpe+MVA-SIV immunization regimen described in this report provide the scientific incentive to explore the efficacy of this vaccine regimen against virulent SIV exposure in the infant macaque model.

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.

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.

Mucosal innate immune response associated with a timely humoral immune response and slower disease progression after oral transmission of simian immunodeficiency virus to rhesus macaques.

Mucosal transmission is the predominant mode of human immunodeficiency virus (HIV) infection worldwide, and the mucosal innate interferon response represents an important component of the earliest host response to the infection. Our goal here was to assess the changes in mRNA expression of innate mucosal genes after oral simian immunodeficiency virus (SIV) inoculation of rhesus macaques (Macaca mulatta) that were followed throughout their course of disease progression. The SIV plasma viral load was highest in the macaque that progressed rapidly to simian AIDS (99 days) and lowest in the macaque that progressed more slowly (>700 days). The mRNA levels of six innate/effector genes in the oral mucosa indicated that slower disease progression was associated with increased expression of these genes. This distinction was most evident when comparing the slowest-progressing macaque to the intermediate and rapid progressors. Expression levels of alpha and gamma interferons, the antiviral interferon-stimulated gene product 2'-5' oligoadenylate synthetase (OAS), and the chemokines CXCL9 and CXCL10 in the slow progressor were elevated at each of the three oral mucosal biopsy time points examined (day 2 to 4, 14 to 21, and day 70 postinfection). In contrast, the more rapidly progressing macaques demonstrated elevated levels of these cytokine/chemokine mRNA at lymph nodes, coincident with decreased levels at the mucosal sites, and a decreased ability to elicit an effective anti-SIV antibody response. These data provide evidence that a robust mucosal innate/effector immune response is beneficial following lentiviral exposure; however, it is likely that the anatomical location and timing of the response need to be coordinated to permit an effective immune response able to delay progression to simian AIDS.

Rapid virus dissemination in infant macaques after oral simian immunodeficiency virus exposure in the presence of local innate immune responses.

A vaccine to protect human immunodeficiency virus (HIV)-exposed infants is an important goal in the global fight against the HIV pandemic. Two major challenges in pediatric HIV vaccine design are the competence of the neonatal/infant immune system in comparison to the adult immune system and the frequent exposure to HIV via breast-feeding. Based on the hypothesis that an effective vaccine needs to elicit antiviral immune responses directly at the site of virus entry, the pattern of virus dissemination in relation to host immune responses was determined in mucosal and lymphoid tissues of infant macaques at 1 week after multiple oral exposures to simian immunodeficiency virus (SIV). The results show that SIV disseminates systemically by 1 week. Infant macaques can respond rapidly to virus challenge and mount strong innate immune responses. However, despite systemic infection, these responses are most pronounced in tissues close to the viral entry site, with the tonsil being the primary site of virus replication and induction of immune responses. Thus, distinct anatomic compartments are characterized by unique cytokine gene expression patterns. Importantly, the early response at mucosal entry sites is dominated by the induction of proinflammatory cytokines, while cytokines with direct antiviral activity, alpha/beta interferons, are only minimally induced. In contrast, both antiviral and proinflammatory cytokines are induced in lymphoid tissues. Thus, although infant macaques can respond quickly to oral viral challenge, the locally elicited immune responses at mucosal entry sites are likely to favor immune activation and thereby virus replication and are insufficient to limit virus replication and dissemination.

Attenuated poxvirus-based simian immunodeficiency virus (SIV) vaccines given in infancy partially protect infant and juvenile macaques against repeated oral challenge with virulent SIV.

An infant macaque model was developed to test pediatric vaccine candidates aimed at reducing HIV transmission through breast-feeding. Infant macaques were given multiple immunizations during the first 3 weeks of life with recombinant poxvirus vaccines expressing simian immunodeficiency virus (SIV) structural proteins Gag, Pol, and Env (ALVAC-SIV or modified vaccinia virus Ankara [MVA]-SIV). After repeated daily oral inoculations with virulent SIVmac251 at 4 weeks of age, significantly fewer ALVAC-SIV-immunized infants were infected compared with unimmunized infants. Monkeys not infected after oral challenge in infancy were rechallenged at 16 months of age or older by repeated weekly oral SIV exposure; unimmunized animals were infected after fewer SIV exposures than were animals vaccinated with ALVAC-SIV or MVA-SIV. When infected, ALVAC-SIV- and MVA-SIV-vaccinated animals also had reduced viremia compared with unimmunized animals. The results of these investigations suggest that immunization of human infants with poxvirus-based HIV vaccine candidates may offer protection against early and late HIV infection through breastfeeding.