Intranasal nanoemulsion-adjuvanted HSV-2 subunit vaccine is effective as a prophylactic and therapeutic vaccine using the guinea pig model of genital herpes.

Genital herpes is a sexually transmitted disease representing a major global health concern. Currently, there is no approved vaccine and existing antiviral therapies exhibit limited efficacy. Herein, we describe an intranasal (IN) vaccine comprised of HSV-2 surface glycoproteins gD2 and gB2 formulated in a nanoemulsion adjuvant (NE01-gD2/gB2). Using the HSV-2 genital herpes guinea pig model, we demonstrate that IN NE01-gD2/gB2 induces higher levels of neutralizing antibody compared to a monovalent IN NE01-gD2 vaccine, but less than an intramuscular (IM) Alum/MPL-gD2 vaccine. Following intravaginal (IVag) challenge with HSV-2, the group immunized with IN NE01-gD2/gB2 exhibited significantly reduced acute and recurrent disease scores compared to placebo recipients. Significantly, latent virus was only detected in the dorsal root ganglia of 1 of 12 IN NE01-gD2/gB2-vaccinated animals compared to 11 of 12 placebo recipient. In the therapeutic model, IN NE01-gD2/gB2 immunized guinea pigs exhibited a significant reduction in the recurrent lesions scores (64%, p < 0.01), number of animal days with disease (64%, p < 0.01), number of animals with viral shedding (50%, p < 0.04) and reduction in virus positive vaginal swabs (56%, p < 0.04), These data suggests that the treatment may be effective in treating chronic disease and minimizing virus transmission. These results warrant advancing the development of IN NE01-gD2/gB2 as both a prophylactic and therapeutic vaccine against HSV-2.

Successful application of prime and pull strategy for a therapeutic HSV vaccine.

One promising approach for a herpes simplex virus vaccine uses a vaccine to prime and a chemoattractant to pull immune cells into the genital tract. We evaluated subunit vaccines (prime) and imiquimod (pull) in the guinea pig (gp) model of recurrent Herpes simplex virus type-2 (HSV-2). Following vaginal HSV-2 infection, gps were vaccinated with various combination of glycoproteins and adjuvant with or without subcutaneous or local applications of imiquimod after infection. Animals were examined daily for recurrent lesions and vaginal swabs collected for recurrent shedding. Although both the vaccines alone and imiquimod alone reduced recurrent HSV disease, the combination of local imiquimod and vaccine (Prime and Pull) was the most effective. In the first study, immunization with the trivalent vaccine alone or imiquimod alone decreased recurrent disease. However, the largest decrease was with the combination of vaccine and local imiquimod (P < 0.001 vs. placebo or vaccine alone). No effect on recurrent shedding was observed. In the second study, recurrent disease scores were similar in the PBS control group and the trivalent-immunized group treated with subcutaneous imiquimod however, significant reductions with glycoprotein vaccines and local imiquimod (p < 0.01 vs. placebo) were noted. The number of qPCR-positive recurrent swabs, ranged from 5 to 11% in the vaccinated+local imiquimod groups compared 29% in the PBS control group (P < 0.05). No recurrent swab samples from vaccinated groups were culture positive. We conclude that the strategy of prime (subunit HSV vaccine) and topical pull (intravaginal/topical imiquimod) decreased recurrent HSV more effectively than vaccine alone.

Duration of protection from live attenuated vs. sub unit HSV-2 vaccines in the guinea pig model of genital herpes: Reassessing efficacy using endpoints from clinical trials.

BACKGROUND: Although herpes simplex viruses (HSV) are a major target for vaccine development no vaccine is currently licensed. METHODS: A live attenuated HSV virus vaccine, VC2 was compared to a subunit HSV vaccine, glycoprotein D (gD2) administered with the adjuvant, MPL/Alum using the guinea pig model of genital herpes. Three doses of intramuscular (IM) vaccine were provided followed by intravaginal challenge with HSV-2 at either 3 weeks or six months after the last vaccination. RESULTS: Both VC2 and gD2 vaccines reduced acute genital disease. VC2 was somewhat more effective in reducing acute vaginal replication, the amount of virus in neural tissue, subsequent recurrent disease and recurrent virus shedding following challenge at 3 weeks post vaccination. Both vaccines continued to provide protection at 6 months after vaccination but the differences between the vaccines became more pronounced in favor of the live attenuated vaccine, VC2. Significant differences in acute disease, acute vaginal virus replication, recurrent disease and recurrent virus shedding (P<0.05 for each) was observed comparing the vaccines. Re-examination of protection for this study using criteria similar to those used in recent clinical trials (inclusion of recurrent disease) showed that efficacy may not be as high in this model as previously thought prompting a need to assess the best predictive outcomes for protection in humans. CONCLUSION: While both the live attenuated vaccine, VC2, and the gD2 subunit vaccine provided protection, the duration of protection appeared to be greater for VC2. Using the same evaluation criteria as used in human trials provided unique insights into the utility of the guinea pig model.

The HSV-1 live attenuated VC2 vaccine provides protection against HSV-2 genital infection in the guinea pig model of genital herpes.

BACKGROUND: Although development of an HSV vaccine is a priority there is currently no vaccine available. The recent failure of subunit vaccines suggest that presentation of more antigens via a live attenuated vaccine may be required for protection. We therefore evaluated VC2, a live attenuated HSV vaccine, engineered to be unable to enter into neuronal axons. METHODS: VC2 pathogenesis was first evaluated in guinea pigs following intravaginal inoculation. VC2 was then evaluated as a prophylactic and therapeutic vaccine and compared protection to a gD2 vaccine adjuvanted with MPL/Alum in the guinea pig model of genital HSV-2. The guinea pig model allows evaluation of acute and recurrent disease, as well as vaginal shedding acutely and during episodes of recurrent activation. RESULTS: VC2 was significantly attenuated in guinea pigs compared to the wild type strain, 17syn+. It replicated poorly at the inoculation site, did not produce any genital disease and rarely infected the neural tissue. After prophylactic vaccination, the VC2 vaccine decreased the clinical severity of acute and recurrent HSV-2 disease and shedding and decreased the quantity of virus in the DRGs. When compared to gD2+MPL/Alum, VC2 was somewhat more effective especially as it relates to neural tissue infection. VC2 was not effective as a therapeutic vaccine. CONCLUSION: The live attenuated prophylactic HSV vaccine, VC2, was effective in the guinea pig model of genital HSV-2. Its decreased ability to infect neural tissues provides advantages over other live attenuated vaccines.

Replication-defective lymphocytic choriomeningitis virus vectors expressing guinea pig cytomegalovirus gB and pp65 homologs are protective against congenital guinea pig cytomegalovirus infection.

BACKGROUND: Congenital cytomegalovirus infection can be life-threatening and often results in significant developmental deficits and/or hearing loss. Thus, there is a critical need for an effective anti-CMV vaccine. OBJECTIVE: To determine the efficacy of replication-defective lymphocytic choriomeningitis virus (rLCMV) vectors expressing the guinea pig CMV (GPCMV) antigens, gB and pp65, in the guinea pig model of congenital CMV infection. METHODS: Female Hartley strain guinea pigs were divided into three groups: Buffer control group (n = 9), rLCMV-gB group (n = 11), and rLCMV-pp65 (n = 11). The vaccines were administered three times IM at 1.54 × 10(6)FFU per dose at 21-day intervals. At two weeks after vaccination, the female guinea pigs underwent breeding. Pregnant guinea pigs were challenged SQ at ∼ 45-55 days of gestation with 1 × 10(5)PFU of GPCMV. Viremia in the dams, pup survival, weights of pups at delivery, and viral load in both dam and pup tissues were determined. RESULTS: Pup survival was significantly increased in the LCMV-gB vaccine group. There was 23% pup mortality in the gB vaccine group (p = 0.044) and 26% pup mortality in the pp65 vaccine group (p = 0.054) compared to 49% control pup mortality. The gB vaccine induced high levels of gB binding and detectable neutralizing antibodies, reduced dam viremia, and significantly reduced viral load in dam tissues compared to control dams (p < 0.03). Reduced viral load and transmission in pups born to gB-vaccinated dams was observed compared to pups from pp65-vaccinated or control dams. CONCLUSIONS: The rLCMV-gB vaccine significantly improved pup survival and also increased pup weights and gestation time. The gB vaccine was also more effective at decreasing viral load in dams and pups and limiting congenital transmission. Thus, rLCMV vectors that express CMV antigens may be an effective vaccine strategy for congenital CMV infection.

Transcriptional repression by the HDAC4-RelB-p52 complex regulates multiple myeloma survival and growth.

Although transcriptional activation by NF-κB is well appreciated, physiological importance of transcriptional repression by NF-κB in cancer has remained elusive. Here we show that an HDAC4-RelB-p52 complex maintains repressive chromatin around proapoptotic genes Bim and BMF and regulates multiple myeloma (MM) survival and growth. Disruption of RelB-HDAC4 complex by a HDAC4-mimetic polypeptide blocks MM growth. RelB-p52 also represses BMF translation by regulating miR-221 expression. While the NIK-dependent activation of RelB-p52 in MM has been reported, we show that regardless of the activation status of NIK and the oncogenic events that cause plasma cell malignancy, several genetically diverse MM cells including Bortezomib-resistant MM cells are addicted to RelB-p52 for survival. Importantly, RelB is constitutively phosphorylated in MM and ERK1 is a RelB kinase. Phospho-RelB remains largely nuclear and is essential for Bim repression. Thus, ERK1-dependent regulation of nuclear RelB is critical for MM survival and explains the NIK-independent role of RelB in MM.

Efficacy of N-methanocarbathymidine against genital herpes simplex virus type 2 shedding and infection in guinea pigs.

BACKGROUND: Current approved nucleoside therapies for genital herpes simplex virus (HSV) infections are effective but improved therapies are needed for treatment of both acute and recurrent diseases. METHODS: The effects of N-methanocarbathymidine were evaluated and compared to acyclovir using guinea pig models of acute and recurrent infection. For acute disease following intravaginal inoculation of 10(6 )pfu HSV-2 (MS strain), animals were treated intraperitoneally beginning 24 h post-infection, and the effects on disease severity, vaginal virus replication, subsequent recurrences, and latent virus loads were evaluated. For evaluation of recurrent infection, animals were treated for 21 days beginning 14 days after infection and disease recurrence and recurrent shedding were evaluated. RESULTS: Treatment of the acute disease with N-methanocarbathymidine significantly reduced the severity of acute disease and decreased acute vaginal virus shedding more effectively than acyclovir. Significantly, none of the animals developed visible disease in the high-dose N-methanocarbathymidine group and this was the only group in which the number of days with recurrent virus shedding was reduced. Treatment of recurrent disease was equivalent to acyclovir when acyclovir was continuously supplied in the drinking water. CONCLUSION: N-methanocarbathymidine was effective as therapy for acute and recurrent genital HSV-2 disease in the guinea pig models.

Knockdown of the Inhibitor of Apoptosis BRUCE Sensitizes Resistant Breast Cancer Cells to Chemotherapeutic Agents.

BACKGROUND AND OBJECTIVES: Management of patients with breast cancer often fails because of inherent or acquired resistance to chemotherapy. BRUCE (BIR repeat containing ubiquitin-conjugating enzyme) is a member of the inhibitor of apoptosis protein (IAP) family. It has various cellular functions including suppression of apoptosis and promotion of cytokinesis. Furthermore, it pays a critical role in promotion of DNA damage repair and preservation of genome stability, a new function recently reported by our group. Although BRUCE is expressed in breast cancer cell lines, its expression in human primary breast tumors and its contribution to chemoresistance in breast cancers has not been explored. Chemotherapeutic drugs are used in the treatment of breast cancer patients. However, they are not effective to all patients and patients often develop resistance. Consequently we explored if BRUCE protein level, as judged by immunohistochemistry (IHC), is higher in primary breast tumors than normal breast tissue. We also examined if depletion of BRUCE, using a lentiviral shRNA approach, enhances cell sensitivity to multiple chemotherapeutic agents, including cisplatin, an agent that induces DNA damage by generating DNA cross-links, and taxol, a microtubule stabilizer and mitotic inhibitor. The reason for including these two chemotherapeutic agents in this study is that they hit two essential cellular processes of DNA repair and cytokinesis in which BRUCE plays critical roles. RESULTS AND METHODS: IHC analysis of BRUCE revealed significantly higher levels of BRUCE in primary breast tumors than normal breast tissue. Knockdown of BRUCE protein expression by lentiviral shRNA resulted in increased sensitivity to cisplatin in the resistant breast cancer MDB-MD-231 cell line. Moreover, depletion of BRUCE in this cell line achieved a more profound level of cell killing when coupled to low doses of cisplatin and taxol combined, rather than either drug used alone. CONCLUSIONS: Our data suggest that elevated protein levels of BRUCE in breast tumors may contribute to chemoresistance in breast cancer patients. In support of this suggestion, our data demonstrate that a reduction in BRUCE expression in breast cancer cell lines increases the toxicity of several chemotherapeutic agents. In all likelihood, the contribution of increased BRUCE levels to chemoresistance are likely due to its roles in suppression of apoptosis, promotion of cytokinesis and facilitation of DNA damage repair. These observations suggest that therapeutic suppression of BRUCE could improve chemosensitivity in chemo-resistant breast cancer patients. Therefore, future development of effective inhibitors of BRUCE could benefit patients with high BRUCE expression and chemoresistance.