An HSV-2 nucleoside-modified mRNA genital herpes vaccine containing glycoproteins gC, gD, and gE protects mice against HSV-1 genital lesions and latent infection.

HSV-1 causes 50% of first-time genital herpes infections in resource-rich countries and affects 190 million people worldwide. A prophylactic herpes vaccine is needed to protect against genital infections by both HSV-1 and HSV-2. Previously our laboratory developed a trivalent vaccine that targets glycoproteins C, D, and E present on the HSV-2 virion. We reported that this vaccine protects animals from genital disease and recurrent virus shedding following lethal HSV-2 challenge. Importantly the vaccine also generates cross-reactive antibodies that neutralize HSV-1, suggesting it may provide protection against HSV-1 infection. Here we compared the efficacy of this vaccine delivered as protein or nucleoside-modified mRNA immunogens against vaginal HSV-1 infection in mice. Both the protein and mRNA vaccines protected mice from HSV-1 disease; however, the mRNA vaccine provided better protection as measured by lower vaginal virus titers post-infection. In a second experiment, we compared protection provided by the mRNA vaccine against intravaginal challenge with HSV-1 or HSV-2. Vaccinated mice were totally protected against death, genital disease and infection of dorsal root ganglia caused by both viruses, but somewhat better protected against vaginal titers after HSV-2 infection. Overall, in the two experiments, the mRNA vaccine prevented death and genital disease in 54/54 (100%) mice infected with HSV-1 and 20/20 (100%) with HSV-2, and prevented HSV DNA from reaching the dorsal root ganglia, the site of virus latency, in 29/30 (97%) mice infected with HSV-1 and 10/10 (100%) with HSV-2. We consider the HSV-2 trivalent mRNA vaccine to be a promising candidate for clinical trials for prevention of both HSV-1 and HSV-2 genital herpes.

Vaccine-induced antibodies to herpes simplex virus glycoprotein D epitopes involved in virus entry and cell-to-cell spread correlate with protection against genital disease in guinea pigs.

Herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) subunit antigen is included in many preclinical candidate vaccines. The rationale for including gD2 is to produce antibodies that block crucial gD2 epitopes involved in virus entry and cell-to-cell spread. HSV-2 gD2 was the only antigen in the Herpevac Trial for Women that protected against HSV-1 genital infection but not HSV-2. In that trial, a correlation was detected between gD2 ELISA titers and protection against HSV-1, supporting the importance of antibodies. A possible explanation for the lack of protection against HSV-2 was that HSV-2 neutralization titers were low, four-fold lower than to HSV-1. Here, we evaluated neutralization titers and epitope-specific antibody responses to crucial gD2 epitopes involved in virus entry and cell-to-cell spread as correlates of immune protection against genital lesions in immunized guinea pigs. We detected a strong correlation between neutralizing antibodies and protection against genital disease. We used a high throughput biosensor competition assay to measure epitope-specific responses to seven crucial gD2 linear and conformational epitopes involved in virus entry and spread. Some animals produced antibodies to most crucial epitopes while others produced antibodies to few. The number of epitopes recognized by guinea pig immune serum correlated with protection against genital lesions. We confirmed the importance of antibodies to each crucial epitope using monoclonal antibody passive transfer that improved survival and reduced genital disease in mice after HSV-2 genital challenge. We re-evaluated our prior study of epitope-specific antibody responses in women in the Herpevac Trial. Humans produced antibodies that blocked significantly fewer crucial gD2 epitopes than guinea pigs, and antibody responses in humans to some linear epitopes were virtually absent. Neutralizing antibody titers and epitope-specific antibody responses are important immune parameters to evaluate in future Phase I/II prophylactic human vaccine trials that contain gD2 antigen.

An HSV-2 Trivalent Vaccine Is Immunogenic in Rhesus Macaques and Highly Efficacious in Guinea Pigs.

A genital herpes vaccine is urgently needed to prevent pain and suffering, reduce the incidence of neonatal herpes, and decrease the risk of HIV acquisition and transmission that accompanies genital infection. We evaluated a trivalent HSV-2 subunit antigen vaccine administered with CpG and alum in rhesus macaques and guinea pigs. The vaccine contains glycoproteins C, D and E (gC2, gD2, gE2) to block virus entry by gD2 and immune evasion by gC2 and gE2. In rhesus macaques, the trivalent vaccine induced plasma and mucosa neutralizing antibodies, antibodies that block gC2 and gE2 immune evasion activities, and stimulated CD4 T cell responses. After intravaginal challenge, a self-limited vaginal infection of brief duration was detected by histopathology and immunohistochemistry in naïve, but not in trivalent immunized macaques. Vaccine efficacy was evaluated in female guinea pigs. Animals were mock immunized, or immunized with gD2, the trivalent vaccine or the trivalent vaccine followed by a booster dose of gD2 (trivalent + gD2). The trivalent and trivalent + gD2 groups were 97% and 99% efficacious, respectively in preventing genital lesions and both outperformed gD2 alone. As a marker of transmission risk, vaginal swabs were evaluated daily for HSV-2 DNA and replication competent virus between five and seven weeks after challenge. HSV-2 DNA shedding was reduced in all groups compared with mock. Shedding of replication competent virus occurred on fewer days in the trivalent than gD2 immunized animals while the trivalent + gD2 group had no shedding of replication competent virus. Overall, the trivalent group had genital lesions on < 1% days and shedding of replication competent virus on 0.2% days. The vaccine has outstanding potential for prevention of genital herpes in humans.

A Trivalent HSV-2 gC2, gD2, gE2 Nucleoside-Modified mRNA-LNP Vaccine Provides Outstanding Protection in Mice against Genital and Non-Genital HSV-1 Infection, Comparable to the Same Antigens Derived from HSV-1.

HSV-1 disease is a significant public health burden causing orofacial, genital, cornea, and brain infection. We previously reported that a trivalent HSV-2 gC2, gD2, gE2 nucleoside-modified mRNA-lipid nanoparticle (LNP) vaccine provides excellent protection against vaginal HSV-1 infection in mice. Here, we evaluated whether this HSV-2 gC2, gD2, gE2 vaccine is as effective as a similar HSV-1 mRNA LNP vaccine containing gC1, gD1, and gE1 in the murine lip and genital infection models. Mice were immunized twice with a total mRNA dose of 1 or 10 µg. The two vaccines produced comparable HSV-1 neutralizing antibody titers, and surprisingly, the HSV-2 vaccine stimulated more potent CD8+ T-cell responses to gE1 peptides than the HSV-1 vaccine. Both vaccines provided complete protection from clinical disease in the lip model, while in the genital model, both vaccines prevented death and genital disease, but the HSV-1 vaccine reduced day two vaginal titers slightly better at the 1 µg dose. Both vaccines prevented HSV-1 DNA from reaching the trigeminal or dorsal root ganglia to a similar extent. We conclude that the trivalent HSV-2 mRNA vaccine provides outstanding protection against HSV-1 challenge at two sites and may serve as a universal prophylactic vaccine for HSV-1 and HSV-2.

Novel Adjuvant S-540956 Targets Lymph Nodes and Reduces Genital Recurrences and Vaginal Shedding of HSV-2 DNA When Administered with HSV-2 Glycoprotein D as a Therapeutic Vaccine in Guinea Pigs.

Herpes simplex virus type 2 (HSV-2) is a leading cause of genital ulcer disease and a major risk factor for acquisition and transmission of HIV. Frequent recurrent genital lesions and concerns about transmitting infection to intimate partners affect the quality of life of infected individuals. Therapeutic vaccines are urgently needed to reduce the frequency of genital lesions and transmission. S-540956 is a novel vaccine adjuvant that contains CpG oligonucleotide ODN2006 annealed to its complementary sequence and conjugated to a lipid that targets the adjuvant to lymph nodes. Our primary goal was to compare S-540956 administered with HSV-2 glycoprotein D (gD2) with no treatment in a guinea pig model of recurrent genital herpes (studies 1 and 2). Our secondary goals were to compare S-540956 with oligonucleotide ODN2006 (study1) or glucopyranosyl lipid A in a stable oil-in-water nano-emulsion (GLA-SE) (study 2). gD2/S-540956 reduced the number of days with recurrent genital lesions by 56%, vaginal shedding of HSV-2 DNA by 49%, and both combined by 54% compared to PBS, and was more efficacious than the two other adjuvants. Our results indicate that S-540956 has great potential as an adjuvant for a therapeutic vaccine for genital herpes, and merits further evaluation with the addition of potent T cell immunogens.

Antibodies to Crucial Epitopes on HSV-2 Glycoprotein D as a Guide to Dosing an mRNA Genital Herpes Vaccine.

The toxicity of mRNA-lipid nanoparticle (LNP) vaccines depends on the total mRNA-LNP dose. We established that the maximum tolerated dose of our trivalent mRNA-LNP genital herpes vaccine was 10 µg/immunization in mice. We then evaluated one of the mRNAs, gD2 mRNA-LNP, to determine how much of the 10 µg total dose to assign to this immunogen. We immunized mice with 0.3, 1.0, 3.0, or 10 µg of gD2 mRNA-LNP and measured serum IgG ELISA, neutralizing antibodies, and antibodies to six crucial gD2 epitopes involved in virus entry and spread. Antibodies to crucial gD2 epitopes peaked at 1 µg, while ELISA and neutralizing titers continued to increase at higher doses. The epitope results suggested no immunologic benefit above 1 µg of gD2 mRNA-LNP, while ELISA and neutralizing titers indicated higher doses may be useful. We challenged the gD2 mRNA-immunized mice intravaginally with HSV-2. The 1-µg dose provided total protection, confirming the epitope studies, and supported assigning less than one-third of the trivalent vaccine maximum dose of 10 µg to gD2 mRNA-LNP. Epitope mapping as performed in mice can also be accomplished in phase 1 human trials to help select the optimum dose of each immunogen in a multivalent vaccine.

Guinea Pig and Mouse Models for Genital Herpes Infection.

This article describes procedures for two preclinical animal models for genital herpes infection. The guinea pig model shares many features of genital herpes in humans, including a natural route of inoculation, self-limiting primary vulvovaginitis, spontaneous recurrences, symptomatic and subclinical shedding of HSV-2, and latent infection of the associated sensory ganglia (lumbosacral dorsal root ganglia, DRG). Many humoral and cytokine responses to HSV-2 infection in the guinea pig have been characterized; however, due to the limited availability of immunological reagents, assessments of cellular immune responses are lacking. In contrast, the mouse model has been important in assessing cellular immune responses to herpes infection. Both the mouse and guinea pig models have been extremely useful for evaluating preventative and immunotherapeutic approaches for controlling HSV infection and recurrent disease. In this article, we describe procedures for infecting guinea pigs and mice with HSV-2, scoring subsequent genital disease, and measuring replicating virus to confirm infection. We also provide detailed protocols for dissecting and isolating DRG (the site of HSV-2 latency), quantifying HSV-2 genomic copies in DRG, and assessing symptomatic and subclinical shedding of HSV-2 in the vagina. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Primary and recurrent genital herpes infection in the guinea pig model Support Protocol 1: Blood collection via lateral saphenous vein or by cardiac puncture after euthanasia Support Protocol 2: Dissection and isolation of dorsal root ganglia from guinea pigs Support Protocol 3: PCR amplification and quantification of HSV-2 genomic DNA from samples Basic Protocol 2: Primary genital herpes infection in the mouse model Alternate Protocol: Flank infection with HSV-2 in the mouse model Support Protocol 4: Dissection and isolation of mouse dorsal root ganglia.

Techniques for Characterizing Cytomegalovirus-Encoded miRNAs.

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level by binding to sites within the 3' untranslated regions of messenger RNA (mRNA) transcripts. The discovery of this completely new mechanism of gene regulation necessitated the development of a variety of techniques to further characterize miRNAs, their expression, and function. In this chapter, we will discuss techniques currently used in the miRNA field to detect, express and inhibit miRNAs, as well as methods used to identify and validate their targets, specifically with respect to the miRNAs encoded by human cytomegalovirus.

Trivalent nucleoside-modified mRNA vaccine yields durable memory B cell protection against genital herpes in preclinical models.

Nucleoside-modified mRNA vaccines have gained global attention because of COVID-19. We evaluated a similar vaccine approach for preventing a chronic, latent genital infection rather than an acute respiratory infection. We used animal models to compare an HSV-2 trivalent nucleoside-modified mRNA vaccine with the same antigens prepared as proteins, with an emphasis on antigen-specific memory B cell responses and immune correlates of protection. In guinea pigs, serum neutralizing-antibody titers were higher at 1 month and declined far less by 8 months in mRNA- compared with protein-immunized animals. Both vaccines protected against death and genital lesions when infected 1 month after immunization; however, protection was more durable in the mRNA group compared with the protein group when infected after 8 months, an interval representing greater than 15% of the animal's lifespan. Serum and vaginal neutralizing-antibody titers correlated with protection against infection, as measured by genital lesions and vaginal virus titers 2 days after infection. In mice, the mRNA vaccine generated more antigen-specific memory B cells than the protein vaccine at early times after immunization that persisted for up to 1 year. High neutralizing titers and robust B cell immune memory likely explain the more durable protection by the HSV-2 mRNA vaccine.

Vaccines to prevent genital herpes.

Genital herpes increases the risk of acquiring and transmitting Human Immunodeficiency Virus (HIV), is a source of anxiety for many about transmitting infection to intimate partners, and is life-threatening to newborns. A vaccine that prevents genital herpes infection is a high public health priority. An ideal vaccine will prevent both genital lesions and asymptomatic subclinical infection to reduce the risk of inadvertent transmission to partners, will be effective against genital herpes caused by herpes simplex virus types 1 and 2 (HSV-1, HSV-2), and will protect against neonatal herpes. Three phase 3 human trials were performed over the past 20 years that used HSV-2 glycoproteins essential for virus entry as immunogens. None achieved its primary endpoint, although each was partially successful in either delaying onset of infection or protecting a subset of female subjects that were HSV-1 and HSV-2 uninfected against HSV-1 genital infection. The success of future vaccine candidates may depend on improving the predictive value of animal models by requiring vaccines to achieve near-perfect protection in these models and by using the models to better define immune correlates of protection. Many vaccine candidates are under development, including DNA, modified mRNA, protein subunit, killed virus, and attenuated live virus vaccines. Lessons learned from prior vaccine studies and select candidate vaccines are discussed, including a trivalent nucleoside-modified mRNA vaccine that our laboratory is pursuing. We are optimistic that an effective vaccine for prevention of genital herpes will emerge in this decade.

Herpes simplex virus type 2 trivalent protein vaccine containing glycoproteins C, D and E protects guinea pigs against HSV-1 genital infection.

A vaccine to prevent genital herpes is an unmet public health need. We previously reported that a trivalent vaccine containing herpes simplex virus type 2 (HSV-2) glycoproteins C, D, and E (gC2, gD2, gE2) produced in baculovirus and administered with CpG/alum as adjuvants blocks immune evasion mediated by gC2 and gE2 and virus entry by gD2. The vaccine protected guinea pigs against HSV-2 vaginal infection. We evaluated whether the HSV-2 vaccine cross-protects against HSV-1 because many first-time genital herpes infections are now caused by HSV-1. Guinea pigs were mock immunized or immunized with the trivalent vaccine and challenged intravaginally with a different HSV-1 isolate in two experiments. Guinea pigs immunized with the trivalent vaccine developed genital lesions on fewer days than the mock group: 2/477 (0.4%) days compared to 15/424 (3.5%) in experiment one, and 0/135 days compared to 17/135 (12.6%) in experiment two (both P < .001). No animal in the trivalent group had HSV-2 DNA detected in vaginal secretions: 0/180 days for trivalent compared to 4/160 (2.5%) for mock (P < .05) in experiment one, and 0/65 days for trivalent compared to 4/65 (6%) for mock in experiment two. Therefore, a vaccine designed to prevent HSV-2 also protects against HSV-1 genital infection.

Blocking antibody access to neutralizing domains on glycoproteins involved in entry as a novel mechanism of immune evasion by herpes simplex virus type 1 glycoproteins C and E.

Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) blocks complement activation, and glycoprotein E (gE) interferes with IgG Fc-mediated activities. While evaluating gC- and gE-mediated immune evasion in human immunodeficiency virus (HIV)-HSV-1-coinfected subjects, we noted that antibody alone was more effective at neutralizing a strain with mutations in gC and gE (gC/gE) than a wild-type (WT) virus. This result was unexpected since gC and gE are postulated to interfere with complement-mediated neutralization. We used pooled human immunoglobulin G (IgG) from HIV-negative donors to confirm the results and evaluated mechanisms of the enhanced antibody neutralization. We demonstrated that differences in antibody neutralization cannot be attributed to the concentrations of HSV-1 glycoproteins on the two viruses or to the absence of an IgG Fc receptor on the gC/gE mutant virus or to enhanced neutralization of the mutant virus by antibodies that target only gB, gD, or gH/gL, which are the glycoproteins involved in virus entry. Since sera from HIV-infected subjects and pooled human IgG contain antibodies against multiple glycoproteins, we determined whether differences in neutralization become apparent when antibodies to gB, gD, or gH/gL are used in combination. Neutralization of the gC/gE mutant was greatly increased compared that of WT virus when any two of the antibodies against gB, gD, or gH/gL were used in combination. These results suggest that gC and gE on WT virus provide a shield against neutralizing antibodies that interfere with gB-gD, gB-gH/gL, or gD-gH/gL interactions and that one function of virus neutralization is to prevent interactions between these glycoproteins.

A trivalent gC2/gD2/gE2 vaccine for herpes simplex virus generates antibody responses that block immune evasion domains on gC2 better than natural infection.

Vaccines for prevention and treatment of genital herpes are high public health priorities. Our approach towards vaccine development is to focus on blocking virus entry mediated by herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) and to prevent the virus from evading complement and antibody attack by blocking the immune evasion domains on HSV-2 glycoproteins C (gC2) and E (gE2), respectively. HSV-2 gC2 and gE2 are expressed on the virion envelope and infected cell surface where they are potential targets of antibodies that bind and block their immune evasion activities. We demonstrate that antibodies produced during natural infection in humans or intravaginal inoculation in guinea pigs bind to gC2 but generally fail to block the immune evasion domains on this glycoprotein. In contrast, immunization of naïve or previously HSV-2-infected guinea pigs with gC2 subunit antigen administered with CpG and alum as adjuvants produces antibodies that block domains involved in immune evasion. These results indicate that immune evasion domains on gC2 are weak antigens during infection, yet when used as vaccine immunogens with adjuvants the antigens produce antibodies that block immune evasion domains.

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

The goals of a genital herpes vaccine are to prevent painful genital lesions and reduce or eliminate subclinical infection that risks transmission to partners and newborns. We evaluated a trivalent glycoprotein vaccine containing herpes simplex virus type 2 (HSV-2) entry molecule glycoprotein D (gD2) and two immune evasion molecules: glycoprotein C (gC2), which binds complement C3b, and glycoprotein E (gE2), which blocks immunoglobulin G (IgG) Fc activities. The trivalent vaccine was administered as baculovirus proteins with CpG and alum, or the identical amino acids were expressed using nucleoside-modified mRNA in lipid nanoparticles (LNPs). Both formulations completely prevented genital lesions in mice and guinea pigs. Differences emerged when evaluating subclinical infection. The trivalent protein vaccine prevented dorsal root ganglia infection, and day 2 and 4 vaginal cultures were negative in 23 of 30 (73%) mice compared with 63 of 64 (98%) in the mRNA group (P = 0.0012). In guinea pigs, 5 of 10 (50%) animals in the trivalent subunit protein group had vaginal shedding of HSV-2 DNA on 19 of 210 (9%) days compared with 2 of 10 (20%) animals in the mRNA group that shed HSV-2 DNA on 5 of 210 (2%) days (P = 0.0052). The trivalent mRNA vaccine was superior to trivalent proteins in stimulating ELISA IgG antibodies, neutralizing antibodies, antibodies that bind to crucial gD2 epitopes involved in entry and cell-to-cell spread, CD4+ T cell responses, and T follicular helper and germinal center B cell responses. The trivalent nucleoside-modified mRNA-LNP vaccine is a promising candidate for human trials.

Antibody responses to crucial functional epitopes as a novel approach to assess immunogenicity of vaccine adjuvants.

We are interested in developing a vaccine that prevents genital herpes. Adjuvants have a major impact on vaccine immunogenicity. We compared two adjuvants, an experimental Merck Sharp & Dohme lipid nanoparticle (LNP) adjuvant, LNP-2, with CpG oligonucleotide combined with alum for immunogenicity in mice when administered with herpes simplex virus type 2 (HSV-2) glycoproteins C, D and E (gC2, gD2, gE2). The immunogens are intended to produce neutralizing antibodies to gC2 and gD2, antibodies to gD2 and gE2 that block cell-to-cell spread, and antibodies to gE2 and gC2 that block immune evasion from antibody and complement, respectively. Overall, CpG/alum was better at producing serum and vaginal IgG binding antibodies, neutralizing antibodies, antibodies that block virus spread from cell-to-cell, and antibodies that block immune evasion domains on gC2. We used a novel high throughput biosensor assay to further assess differences in immunogenicity by mapping antibody responses to seven crucial epitopes on gD2 involved in virus entry or cell-to-cell spread. We found striking differences between CpG/alum and LNP-2. Mice immunized with gD2 CpG/alum produced higher titers of antibodies than LNP-2 to six of seven crucial epitopes and produced antibodies to more crucial epitopes than LNP-2. Measuring epitope-specific antibodies helped to define mechanisms by which CpG/alum outperformed LNP-2 and is a valuable technique to compare adjuvants.

A trivalent subunit antigen glycoprotein vaccine as immunotherapy for genital herpes in the guinea pig genital infection model.

An estimated 417 million people worldwide ages 15 to 49 are infected with herpes simplex virus type 2 (HSV-2), the most common cause of genital ulcer disease. Some individuals experience frequent recurrences of genital lesions, while others only have subclinical infection, yet all risk transmitting infection to their intimate partners. A vaccine was developed that prevents shingles, which is a recurrent infection caused by varicella-zoster virus (VZV), a closely related member of the Herpesviridae family. The success of the VZV vaccine has stimulated renewed interest in a therapeutic vaccine for genital herpes. We have been evaluating a trivalent subunit antigen vaccine for prevention of genital herpes. Here, we assess the trivalent vaccine as immunotherapy in guinea pigs that were previously infected intravaginally with HSV-2. The trivalent vaccine contains HSV-2 glycoproteins C, D, and E (gC2, gD2, gE2) subunit antigens administered with CpG and alum as adjuvants. We previously demonstrated that antibodies to gD2 neutralize the virus while antibodies to gC2 and gE2 block their immune evasion activities, including evading complement attack and inhibiting activities mediated by the IgG Fc domain, respectively. Here, we demonstrate that the trivalent vaccine significantly boosts ELISA titers and neutralizing antibody titers. The trivalent vaccine reduces the frequency of recurrent genital lesions and vaginal shedding of HSV-2 DNA by approximately 50% and almost totally eliminates vaginal shedding of replication-competent virus, suggesting that the trivalent vaccine is a worthy candidate for immunotherapy of genital herpes.

Human Cytomegalovirus MicroRNAs miR-US5-1 and miR-UL112-3p Block Proinflammatory Cytokine Production in Response to NF-κB-Activating Factors through Direct Downregulation of IKKα and IKKß.

Emerging evidence indicates that human cytomegalovirus (HCMV) manipulates host cell signaling pathways using both proteins and noncoding RNAs. Several studies have shown that HCMV induces NF-κB signaling early in infection, resulting in the induction of antiviral proinflammatory cytokines with a subsequent reduction of these cytokines late in infection. The mechanism for late cytokine reduction is unknown. In this study, we show that HCMV microRNAs (miRNAs) miR-US5-1 and miR-UL112-3p target the IκB kinase (IKK) complex components IKKα and IKKß to limit production of proinflammatory cytokines in response to interleukin 1ß (IL-1ß) and tumor necrosis factor alpha (TNF-α). Transfection of miR-UL112-3p and miR-US5-1 mimics reduced endogenous IKKα and IKKß protein levels, and site-directed mutagenesis of the 3' untranslated regions (UTRs) identified the binding sites for each miRNA. Infection with mutant viruses lacking these miRNAs resulted in increased levels of IKKα and IKKß proteins, an impaired ability to control NF-κB signaling at late times of lytic infection, and increased production of proinflammatory cytokines compared to wild-type virus in cell types relevant to HCMV infection in vivo These phenotypes were rescued by preexpression of miR-US5-1 and miR-UL112-3p in infected cells or by a miR-US5-1/miR-UL112-3p double mutant virus that expresses short hairpin RNAs (shRNAs) targeting IKKα and IKKß, demonstrating the gene specificity of the miRNAs. These observations describe a mechanism through which HCMV miRNAs expressed late in the infectious cycle downregulate proinflammatory cytokine production to create a cellular proviral environment.IMPORTANCE Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in transplant recipients and causes hearing loss and mental retardation when acquired congenitally. Initial events during HCMV infection result in the activation of NF-κB signaling, which culminates in the production of IL-6, CCL5, and TNF-α. Several viruses have developed mechanisms to block the antiviral effects of these cytokines. We show here that two HCMV miRNAs, miR-US5-1 and miR-UL112-3p, specifically downregulate IKKα and IKKß signaling factors necessary to propagate NF-κB signaling and subsequent IL-6, CCL5, and TNF-α production. Regulation of these proinflammatory cytokines during lytic infection and during latency is critical to viral survival in the host.

Techniques for characterizing cytomegalovirus-encoded miRNAs.

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level, by binding to sites within the 3' untranslated regions of messenger RNA (mRNA) transcripts. The discovery of this completely new mechanism of gene regulation necessitated the development of a variety of techniques to further characterize miRNAs, their expression, and function. In this chapter, we will discuss techniques currently used in the miRNA field to express, detect, and inhibit miRNAs as well as methods used to identify their targets.

Cytomegalovirus miRNAs target secretory pathway genes to facilitate formation of the virion assembly compartment and reduce cytokine secretion.

Herpesviruses, including human cytomegalovirus (HCMV), encode multiple microRNAs (miRNA) whose targets are just being uncovered. Moreover, miRNA function during the virus life cycle is relatively unknown. We find that HCMV miRs UL112-1, US5-1, and US5-2 target multiple components of the host secretory pathway, including VAMP3, RAB5C, RAB11A, SNAP23, and CDC42. A HCMV miR UL112-1, US5-1, and US5-2 triple mutant displayed aberrant morphogenesis of the virion assembly compartment (VAC), increased secretion of noninfectious particles, and increased IL-6 release from infected cells. Ectopic expression of miRs UL112-1, US5-1, and US5-2 or siRNAs directed against RAB5C, RAB11A, SNAP23, and CDC42 caused the loss of Golgi stacks with reorganization into structures that resemble the VAC and a decrease in cytokine release. These observations indicate that multiple HCMV miRNAs coordinately regulate reorganization of the secretory pathway to control cytokine secretion and facilitate formation of the VAC for efficient infectious virus production.

Herpes simplex virus type 1 and 2 glycoprotein C prevents complement-mediated neutralization induced by natural immunoglobulin M antibody.

Glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) binds complement component C3b and protects virus from complement-mediated neutralization. Differences in complement interacting domains exist between gC of HSV-1 (gC1) and HSV-2 (gC2), since the amino terminus of gC1 blocks complement C5 from binding to C3b, while gC2 fails to interfere with this activity. We previously reported that neutralization of HSV-1 gC-null virus by HSV antibody-negative human serum requires activation of C5 but not of downstream components of the classical complement pathway. In this report, we evaluated whether activation of C5 is sufficient to neutralize HSV-2 gC-null virus, or whether formation of the membrane attack complex by C6 to C9 is required for neutralization. We found that activation of the classical complement pathway up to C5 was sufficient to neutralize HSV-2 gC-null virus by HSV antibody-negative human serum. We evaluated the mechanisms by which complement activation occurred in seronegative human serum. Interestingly, natural immunoglobulin M antibodies bound to virus, which triggered activation of C1q and the classical complement pathway. HSV antibody-negative sera obtained from four individuals differed over an approximately 10-fold range in their potency for complement-mediated virus neutralization. These findings indicate that humans differ in the ability of their innate immune systems to neutralize HSV-1 or HSV-2 gC-null virus and that a critical function of gC1 and gC2 is to prevent C5 activation.