Targeting the Genital Tract Mucosa with a Lipopeptide/Recombinant Adenovirus Prime/Boost Vaccine Induces Potent and Long-Lasting CD8+ T Cell Immunity Against Herpes: Importance of

Targeting the Genital Tract Mucosa with a Lipopeptide/Recombinant Adenovirus Prime/Boost Vaccine Induces Potent and Long-Lasting CD8+ T Cell Immunity Against Herpes: Importance of

Targeting of the mucosal immune system of the genital tract with subunit vaccines has failed to induce potent and durable local CD8(+) T cell immunity, which is crucial for protection against many sexually transmitted viral pathogens, including HSV type 2 (HSV-2), which causes genital herpes. Louis area co-investigator Dr. The lipopeptide vaccine and the rAdv5 vaccine express the immunodominant HSV-2 CD8(+) T cell epitope (gB(498-505)), and both were delivered intravaginally in the progesterone-induced B6 mouse model of genital herpes. There were less than half of the cases of genital herpes caused by HSV-1 — 58 percent fewer — in women who received the investigational vaccine compared to women who received the control vaccine. Bruce Hirsch, MD, also thinks we are far away from an effective vaccine against genital herpes. Most notably, an optimized vaccine formulation of FI-HSV2 MPL/Alhydrogel given i.m. Taken together, these findings indicate that targeting the VM with a Lipo/rAdv5 prime/boost vaccine elicits a potent, MyD88-dependent, and long-lasting mucosal CD8+ T cell protective immunity against sexually transmitted herpes infection and disease.

The “pulling” involved recruitment of activated T cells directly into the vaginal tissue, via topical application, of chemokines — substances that help mobilize the immune cells. The virus can cause severe neurological disease and even death in infants born to women who are infected with HSV and the virus is a risk factor for sexual transmission of HIV. Increasing evidence demonstrates a substantial link between the epidemics of sexually transmitted HIV-1 and HSV-2 infection (12). With HSV, it has already been established that an infection can generate memory T cells that persist in the periphery and provide enhanced immunity {1}. This may play a role in why the results don’t match up. Based on these findings, the authors noted that while the investigational vaccine holds promise, it will not yet protect the general population against genital herpes. Ongoing studies are attempting to explain this perplexing result.

A vaccine that prevents genital herpes infection will have major public health benefits. An emerging and promising mucosal immunization approach to elicit potent CD8+ T cell responses is to use recombinant viruses (16). Several studies have recently used replication-defective adenovirus vectors to induce potent local viral-specific CD8+ T-cells in GT-DLN and to mobilize them quickly into the VM tissues (8, 16, 17). The actual frequency of recurrence varies however the frequency may decrease Herpes Vaccine 2012 over time. Investing includes risks, including loss of principal. peptide and DNA) (reviewed in (18)); (ii) its ability to elicit a considerably potent and long-lasting pathogen-specific CD8+ T cell response in humans (19); and (iii) its natural tropism for mucosal tissues (20–22), which makes it an ideal antigen delivery system for IVAG vaccination. Although some progress has been made in defining the cellular mechanisms of the immunogenicity of lipopeptide and rAdv-based vaccines, relatively little is known about the underlying innate molecular pathways.

Mucosal delivery of lipopeptide and rAdv vaccines potentially recruits many of the thirteen known mammalian Toll-like receptor (TLR) pathways (6). Each TLR has Toll/Interleukin-1 receptor (TIR) domains that engage two main intracellular signaling pathways: (1) the myeloid differentiation factor 88 (MyD88) pathway; and (2) the TIR domain containing adaptor-inducing IFN-beta (TRIF) pathway (23). One hypothesis, Belshe said, is HSV-1 is more easily killed by antibodies than is HSV-2. The results show that: (i) priming with a lipopeptide and boosting with a rAd5 vector, both expressing the same HSV-2gB498-505 epitope and both delivered IVAG, induced a much more potent local mucosal HSV-specific IFN-γ-producing CD8+ T cells than the homologous lipopeptide/lipopeptide (Lipo/Lipo) vaccine (p ≤ 0.05); (ii) Lipo/rAdv5 prime/boost mucosal vaccine elicits long-lasting CD8+ T cells, with a faster kinetic of mobilization, than the Lipo/Lipo vaccine; (iii) the HSV-specific CD8+ T cell responses induced by the Lipo/rAdv5 prime/boost mucosal vaccine are associated with protection against genital herpes infection and disease; (iv) the HSV-specific CD8+ T cell responses induced by the Lipo/rAdv5 prime/boost mucosal vaccine persisted up to 8 months post-immunization; and (v) MyD88 pathway plays a pivotal role in the HSV-specific CD8+ T cell response and protection induced following IVAG immunization with the Lipo/rAdv5 vaccine. Altogether, these findings lay the foundation for an accessible and durable mucosal prime/boost T-cell based vaccine approach to reduce genital herpes, and presumably other sexually transmitted pathogens. MyD88 deficient mice (MyD88(−/−) mice), 4–5 weeks old, were provided by Dr. Shizuo Akira (Osaka University, Osaka, Japan), and were on the C57BL/6 background.

Female C57BL/6 (B6) mice, 4–5 weeks old, were purchased from the Jackson Laboratory (Bar Harbor, ME). Animal studies conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health. Triple plaque-purified 333 HSV-2 was prepared, as we described previously (6). The live attenuated thymidine kinase-deficient HSV-2 (HSV-2 TK(−)), was provided by Dr. James R. Smiley and Dr. The success of these studies led us to evaluate whether blocking immune evasion domains on gC2 improved the effectiveness of a bivalent HSV-2 subunit antigen vaccine consisting of gC2 and gD2.

Morrison. Triple plaque-purified HSV-2 was prepared as we described previously (55). Once the doctor suspects eye herpes he will then Genital Herpes Vaccine 2012 perform a test known as the slit lamp examination. Heat-killed virus was made by heating virus solution at 100°C for 5 min. HSV inactivation was confirmed by the inability to produce plaques when tested on Vero cells, as we described (1). A recombinant adenovirus virus type 5 (rAdv5) expressing the H-2Kb-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), was constructed. Briefly, the DNA sequence (PB1) encoding the H2Kb SSIEFARL CD8+ CTL target peptide (HSV-gB498-505) (30) was synthesized and cloned into pShuttle-CMV (Stratagene, Santa Clara, CA) vector between KpnI and XbaI sites.
Targeting the Genital Tract Mucosa with a Lipopeptide/Recombinant Adenovirus Prime/Boost Vaccine Induces Potent and Long-Lasting CD8+ T Cell Immunity Against Herpes: Importance of

The transfer pShuttle-CMV-PB1 plasmid DNA was linearized with PmeI, gel-purified and then transformed into BJ5183-AD-1 bacterial cells (Stratagene, Santa Clara, CA) carrying the pAdEasy-1 plasmid by electroporation to generate recombinant adenovirus plasmid pAdEasy-1-PB1. The recombinant pAdEasy-1-PB1 plasmid DNA was linearized with PacI and transfected into 293 cells to produce recombinant adenovirus Ad5.CMV-PB1 that will express the HSV-gB498-505 CD8+ T cell epitopes by CMV promoter. The Ad5.CMV-PB1 recombinant adenovirus was plaque-purified and amplified to the titer of 108 PFU/ml. The virus stock was aliquoted and stored in −80°c. The H2Kb SSIEFARL CD8+ CTL target peptide (HSV-gB498-505) (56) and the [dA]K[Cha]VAAWTLKAA[dA][Ahx]C Pan DR peptide (PADRE) (30) were synthesized either individually or as PADRE-CTL chimeric epitopes using Fmoc (9-Fluorenylmethoxycarbonyl) chemistry, with PyBOP/HOBt (Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate/N-hydroxybenzotriazole) activation, as we described previously (30, 57). The parental peptides were washed three times with DMF (dimethylformamide) and treated twice with 2% hydrazine in DMF. After additional washings (x 6) with DMF, hydrazine acetic acid peptides were cleaved using TFA/TIS/H2O (trifluoroacetic acid/tri-isopropylsilane/water; 95:2.5:2.5) and the resultant peptides precipitated in cold ether.

The peptides were then washed (x 3) with cold ether and analyzed by MS and HPLC. Purification of the peptides was performed using Gilson HPLCs, Vydac C18 columns (2.2 × 25 cm or for larger amounts of crude peptide, 5 × 25 cm). The analysis was performed using Vydac C18, 5um, 0.46 × 25 cm columns, with a gradient of 2% per minute of water, 0.1% TFA, 95% acetonitrile and 0.1% TFA. Once peptides were purified to over 95% purity rate, they were lyophilized. Mass spectrometric analysis was performed by MDS/Sciex QStar XL mass spectrometer equipped with a nanospray source. Expression of gC2, gD2, and gE2 subunit antigens in baculovirus (bac).Bac-gC2(426t) (gC2 subunit antigen) and bac-gD2(306t) (gD2 subunit antigen) express gC2 and gD2, respectively, derived from HSV-2 strain 333 and are truncated prior to the transmembrane domain (30, 36–38). The prototype Th-CTL chimeric lipopeptide vaccines were synthesized following, one, two or three N-terminal attachments of glyoxylyl lipid to the Th-CTL backbone using chemoselective ligation as previously described (6).

This was achieved by adding one, two or three lysine residues whose side-chains were selectively protected with ivDde (1-(4,4-Dimethyl-2,6-dioxoxyxlohex-1-ylidene)-3-methylbutyl), a hydrazine-sensitive side-chain protecting group. Additional studies conducted in different communities with high vaccination rates will help elucidate the duration of immunity afforded by varicella vaccination and the roles of both mechanisms of immunologic boosting: periodic exogenous exposures and asymptomatic endogenous reactivations. The synthesis of the glyoxylyl derivative of palmitate and ligation of peptides were performed using a modification of chemoselective ligation (6). Briefly, dimethyl-2,3-O-isopropylidene tartrate was added to a polyethylene glycol amino resin using PyBOP activation. The second ester was then displaced via the addition of 1,3-diaminopropane. Finally palmitic acid was activated using PyBOP and used to acylate the amino terminus. Treatment with TFA followed by periodate oxidation generated the alpha-oxo-aldehyde moiety.

Following lyophilization, the peptides were transferred in 50 mL round bottom flasks, fitted with septa and flushed with nitrogen. A minimal amount of degassed water was added until the peptides were solubilized and displayed as a gel. Stochiometric amounts of lipid were then added in 2-methyl-propan-2-ol drop-wise with stirring. The final ratio of water to organic solvent was 95:5. To add the 2nd and 3rd lipids, an aliquot equal to 120% of the concentration of the peptide was added, with 10–20 minutes of stirring in between each addition. The reactions were monitored using the QStar XL mass spectrometer (). The disappearance of the parent peptide was observed concomitantly with the appearance of the lipid-tailed peptide.

In all cases, the parent peptide was not detectable at the end of the acylation process. All immunizations were carried out with 100 ug of lipopeptide vaccine and 5 × 107 of the rAd5 vaccine, both delivered intravaginally (IVAG) in sterile PBS on day 0 and 21 (, , , , and ). As a negative control mice were primed IVAG with the irrelevant OVA257-264 lipopeptide and boosted with an empty Ad5 vector (mock-immunized mice). As a positive control, mice were inoculated IVAG with 5×103 PFU of the HSV-2 TK(−) virus, as previously described (6, 44) (, , and ). Wells were coated with 1 μg/ml of human or mouse IgG prepared by purification on a protein G column (Pierce), purchased guinea pig IgG (Santa Cruz Biotechnology), or 10% bovine serum albumin (BSA) at 4°C overnight; blocked with 5% milk; and incubated with 200 ng, 400 ng, or 800 ng bac-gE2(24-405t) or bac-gD2(306t) for 1 h at room temperature, and bound gE2 was detected by using R265 anti-gE2 or R122 anti-gD2 serum followed by HRP-conjugated donkey anti-rabbit IgG. Ten days after the final immunization, each group of mice was IP injected daily with 4 doses of 0.5 mg of Depo-Provera in 100 uL sterile PBS. Mice were then challenged intravaginally, on day 14, either with 5 × 106 pfu (= 200 x LD50 for survival analysis) or with 5 × 104 pfu (for virus titers and disease analysis) of HSV-2 (strain 333).

In more-recent years, outbreaks have been reported to occur almost exclusively in elementary schools, some of which have extremely high vaccine coverage (96%–98%) [10, 19, 21, 25]. After overnight fixation the VM samples were cut into small longitudinal bands. Then samples were blocked with anti-FcRg, antibody (US biological, MA) at a dilution of 1:100 and in goat serum/PBS overnight. The anti-CD8 antibody conjugated to FITC at a dilution of 1:100 and 14.3 mM DAPI (Molecular probes, Invitrogen, CA) were applied overnight at 4°C. Then samples were mounted in 50% glycerol/PBS. Confocal microscopy was performed with a laser confocal and multiphoton microscope system with a conventional laser confocal microscope (Zeiss LSM 510 META, Jena, Germany) equipped with a Femtosecond titanium laser (Chameleon, Coherent, CA). Lymphocytes were isolated from the female genital tract (GT) mucosal tissues and treated with calcium- and magnesium-free phosphate-buffered saline for whole-body perfusion prior to tissue harvest, as previously described (29).

The female GT included the ovaries, fallopian tubes, uterus, and vagina (5, 6). GT tissues were digested following a 2 hrs treatment with 2.5 mg/mL of collagenase type A (Roche catalogue number 1088 785) and 5 units/mL of DNase I (Roche catalogue number 104 159) suspended in RPMI-1640 with 5% fetal bovine serum, penicillin-streptomycin, HEPES. Mucosal GT tissues were pooled from five-mice/immunization group to provide sufficient cells to perform replicates of each experiment (IFN-γ ELISpot) and to allow for accurate measurement of immune responses in each immunization group. The average yield of cells per mouse was 4 × 106 cells/female reproductive tract. Two weeks after the second immunization, mice were euthanized, the iliac and inguinal lymph nodes (5, 6) draining the GT (GT-DLN) were removed and single cell suspensions of GT-DLN cells placed into ice-cold serum free HL-1 medium supplemented with 15 mM HEPES, 5 × 10−5 M β-mercaptoethanol, 2 mM glutamine, 50 IU of penicillin and 50ug of streptomycin (GIBCO-BRL, Grand Island, NY) (referred as complete medium, or CM) (6). The cells were cultured in 6-well plates at 5 × 106 cells/well in CM with the target HSV-gB498-505 alone; with UV inactivated HSV infected stimulator cells at MOI of 3, or with Con-A (positive control). 72 hours later, the supernatants were collected and the concentrations of IFN-γ, TNF-α and IL-2 were determined in a specific sandwich ELISA according to the manufactured instructions (BD PharMingen, San Diego, CA).

You may also like