Contenuto scaricabileScarica il pdf
Prevention of Anogenital Herpes Infection Using Adjuvanted Liposomal GD3PEP Vaccines
Herpes simplex virus (HSV) is the causative agent of anogenital herpes infection, the second most common sexually transmitted infection in the United States. There are two HSV serotypes, HSV-1 and HSV-2, which both can cause anogenital infection and are associated with recurrent infections due to establishment of viral latency in the neuronal ganglia. Infections with HSV-1 or HSV-2 can also cause life-threatening disseminated infections in neonates and immunocompromised individuals. Anti-viral drugs reduce the frequency and severity of symptoms, but can also be toxic and resistant strains of HSV are common in immunocompromised individuals. Currently, no vaccine exists to prevent HSV infections. Studies in our laboratory have focused on using conjugatable adjuvant lipid vesicles (CALV) linked to the HSV antigen, gD3pep, as a vaccine. The gD3 peptide is composed of three immunogenic segments of the HSV-2 surface glycoprotein D. This peptide has a 92.8% protein homology between HSV-1 and HSV-2. We hypothesized that CALV-gD3pep containing a single or combination of immunoenhancing adjuvants would protect female BALB/c mice from a lethal intravaginal (IVa) challenge with HSV-2 or HSV-1. We also established an intrarectal (IR) model of HSV infection in male and female BALB/c mice allowing us to compare the efficacy of adjuvanted CALV-gD3pep vaccines in male and female mice. In study 1, female mice were vaccinated 3X subcutaneously (SC) with gD3pep, CALV unlinked with gD3pep, CALV-gD3pep with no adjuvant, or CALV-gD3pep containing the Pam3CAG, QS-21, or GPI-0100 adjuvant and IVa challenged with HSV-2. In study 2, female mice were vaccinated 3X SC with CALV-gD3pep containing MPL, QS-21, or a combination of the two adjuvants or vaccinated 1X with CALV-gD3pep containing MPL plus collagen or just MPL and IVa challenged with HSV-2. In Study 3, BALB/c mice were IV challenged with HSV-1 (undiluted stock virus, 1:2 dilution, or 1:4 dilution), and the 1:4 dilution of HSV-1 was used to IV challenge BALB/c mice vaccinated SC with CALV-gD3pep containing the adjuvants MPL, MTP-PE, LT1, or CDN during Study 4. In study 5, female mice were IR challenged with a 1:2 or 1:4 dilution of HSV-1 or HSV-2 using 2% nonoxynol-9 to promote viral infection. In study 6, female mice were IR challenged with HSV-1 using 4% nonoxynol-9 and male mice were IR challenged with HSV-1 or HSV-2. In study 7, male and female mice were IR challenged with HSV-2 using 3% nonoxynol-9 and a 20 μL instead of 10 μL viral challenge dose. Control mice were administered PBS. Mice were monitored 2X/day for disease signs. Viral burden was assessed in the vagina and spinal cord using a plaque forming unit assay (PFU) with Vero cells. Blood and spleens were harvested from vaccinated mice to determine serum anti-HSV neutralizing antibody titers, anti-gD3pep IgG1 and IgG2a serum concentrations (ELISA assay), and splenocyte secretion of IL-4 or IFN-γ following incubation with gD3pep (ELISpot assay). A Luminex assay was also used to determine the pro- and anti-inflammatory cytokines produced by the splenocytes. In studies 1 and 2, mice vaccinated with gD3pep alone or PBS had poor survival (14% and 0%, respectively) with HSV-2 IVa challenge. Survival was increased with CALV-gD3pep without adjuvant (29%), and much better with CALV-gD3pep with the QS-21 or GPI-0100 saponin adjuvants (57%), CALV-gD3pep with MPL (86%), and CALV-gD3pep with a combination of MPL and QS-21 (86-100%). CALV-gD3pep containing Pam3CAG also had low survival (29% survival) as did single dose CALV-gD3pep containing MPL, with or without collagen (0% survival). The morbidity and viral burden paralleled the survival results. In Study 4, HSV-1 IVa challenged mice were well protected by CALV-gD3pep containing CDN or MPL (85.7% survival) or MTP-PE (71.4% survival), with decreased disease signs and viral burden compared to control mice or mice vaccinated with CALV-gD3pep containing LT1 (14.3% survival for both). In studies 5-7, both HSV-1 and HSV-2 rectal infections were established in male and female mice using 3% nonoxynol-9 and an increased amount of the HSV challenge dose. Overall, protection against HSV-1 or HSV-2 vaginal infection was associated with a strong Th1 response (serum IgG2a and IFN-γ secreting splenocytes) complemented by anti-inflammatory cytokine (IL-10 and IL-4) secretion. There was also an elevated Th2 response (serum IgG1 and IL-4 secreting splenocytes). These studies showed that CALV-gD3pep vaccines combined with an effective adjuvant can provide significant protection against vaginal herpes infections caused by either HSV-1 or HSV-2 in female BALB/c mice. The protective effects of the adjuvanted CALV-gD3pep vaccines were mainly associated with a Th1-like response, but a Th2-like response was also slightly protective. In addition, an intrarectal HSV-1 or HSV-2 infection can be established in male and female mice and used in future CALV-gD3pep vaccine studies.