Kenneth L. Rosenthal

Intranasal Immunization with CpG Oligodeoxynucleotides as an Adjuvant Dramatically Increases IgA and Protection Against Herpes Simplex Virus-2 in the Genital Tract

Development of vaccines capable of preventing the transmission or limiting the severity of sexually transmitted viruses, such as HSV and HIV, will likely be dependent on the induction of potent long-lasting mucosal immune responses in the genital tract. Recently, synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs were shown to serve as potent adjuvants for the induction of mucosal immune responses. Here, we show that intranasal immunization with CpG ODN, plus recombinant glycoprotein B (rgB) of HSV-1, results in significantly elevated levels of specific anti-gB IgA Abs in vaginal washes that remained high throughout the estrous cycle. Additionally, dramatically elevated numbers of specific IgA Ab-secreting cells were present and persisted in the genital tract in response to intravaginal (IVAG) HSV-2 challenge. HSV-2-specific CTL were observed at moderate levels in the spleens of CpG or non-CpG ODN-immunized mice. In contrast, strong CTL responses were observed locally in the genital tissues of both groups following IVAG HSV-2 challenge. Interestingly, mice immunized intranasally with rgB plus CpG ODN, but not non-CpG ODN, were significantly protected following IVAG HSV-2 challenge. Measurement of virus in protected CpG-immunized mice revealed a log lower level of replication within the first few days after infection. In conclusion, these results indicate that intranasal immunization with CpG ODN plus protein mediates immunity in the female genital tract capable of protecting against a sexually transmitted pathogen.

Interleukin-15 and Natural Killer and NKT Cells Play a Critical Role in Innate Protection against Genital Herpes Simplex Virus Type 2 Infection

ABSTRACT Interleukin-15 (IL-15), natural killer (NK) cells, and NK T (NKT) cells, components of the innate immune system, are known to contribute to defense against pathogens, including viruses. Here we report that IL-15−/− (NK− and NKT−/+) mice and RAG-2−/−/γc−/− (NK− and NKT−) mice that lack all lymphoid cells were very susceptible to vaginal infection with a low dose of herpes simplex virus type 2 (HSV-2). IL-15−/− and RAG-2−/−/γc−/− mice were 100-fold more susceptible and RAG-2−/−, CD-1−/− (NKT−), and gamma interferon (IFN-γ)−/− mice were 10-fold more susceptible to vaginal HSV-2 infection than control C57BL/6 mice. NK and/or NKT cells were the early source of IFN-γ in vaginal secretions following genital HSV-2 infection. This study demonstrates that IL-15 and NK-NKT cells are critical for innate protection against genital HSV-2.

Progesterone Increases Susceptibility and Decreases Immune Responses to Genital Herpes Infection

ABSTRACT Depo-provera, a long-acting progestational formulation, is widely used to facilitate infection of sexually transmitted diseases in animal models. We have previously reported that hormone treatments change susceptibility and immune responses to genital tract infections. In this study we compared the changes in susceptibility of mice to genital herpes simplex virus type 2 (HSV-2) after Depo-provera or a saline suspension of progesterone (P-sal). We found that following Depo-provera-treatment, mice had prolonged diestrus that lasted more than 4 weeks. This coincided with a 100-fold increase in susceptibility to genital HSV-2 compared to that of untreated mice. Mice given P-sal were in diestrous stage for 4 to 6 days before returning to irregular reproductive cycles. When these mice were infected at diestrus they showed a 10-fold increase in susceptibility compared to that of normal, untreated mice. P-sal-treated mice infected at estrus were susceptible to HSV-2, depending on the infectious dose. Normal, untreated mice in estrus were not susceptible to HSV-2, even at a high infectious dose of 107 PFU. In addition to alterations in susceptibility, Depo-provera treatment had inhibitory effects on immune responses to HSV-2. Mice immunized with HSV-2 protein (gB) and treated with Depo-provera showed significant lowering of local HSV-2-specific immunoglobulin G (IgG) and IgA in their vaginal washes. Mice immunized with an attenuated strain of HSV-2 2 weeks after Depo-provera treatment failed to develop protection when challenged intravaginally with wild-type HSV-2. In contrast, mice given progesterone and immunized at diestrus or estrus were completely protected from intravaginal challenge. These studies show that Depo-provera treatment changes susceptibility and local immune responses to genital HSV-2 infection. Animal models and vaccine strategies for sexually transmitted diseases need to consider the effect of hormone treatments on susceptibility and immune responses.

Local Delivery of CpG Oligodeoxynucleotides Induces Rapid Changes in the Genital Mucosa and Inhibits Replication, but Not Entry, of Herpes Simplex Virus Type 2

ABSTRACT Mucosal surfaces are the entry sites for the vast majority of infectious pathogens and provide the first line of defense against infection. In addition to the epithelial barrier, the innate immune system plays a key role in recognizing and rapidly responding to invading pathogens via innate receptors, such as Toll-like receptors (TLR). Bacterial CpG DNA, a potent activator of innate immunity, is recognized by TLR9. Here, we confirm that local mucosal, but not systemic, delivery of CpG oligodeoxynucleotides (ODN) to the genital tract protects mice from a subsequent lethal vaginal herpes simplex virus type 2 (HSV-2) challenge. Since these effects were so local in action, we examined the genital mucosa. Local delivery of CpG ODN induced rapid proliferation and thickening of the genital epithelium and caused significant recruitment of inflammatory cells to the submucosa. Local CpG ODN treatment also resulted in inhibition of HSV-2 replication but had no effect on HSV-2 entry into the genital mucosa. CpG ODN-induced protection against HSV-2 was not associated with early increases in gamma interferon (IFN-γ) secretion in the genital tract, and CpG ODN-treated IFN-γ−/− mice were protected from subsequent challenge with a lethal dose of HSV-2. Treatment of human HEK-293 cells transfected with murine TLR9 showed that the antiviral activity of CpG ODN was mediated through TLR9. These studies suggest that local induction of mucosal innate immunity can provide protection against sexually transmitted infections, such as HSV-2 or possibly human immunodeficiency virus, at the mucosal surfaces.

Specific secretory immune responses in the female genital tract following intranasal immunization with a recombinant adenovirus expressing glycoprotein B of herpes simplex virus

Previously, we demonstrated that intranasal (i.n.) but not intraperitoneal (i.p.) immunization with a recombinant adenovirus vector expressing glycoprotein B (gB) of herpes simplex virus type 1 (HSV-1) induced mucosal immune responses and conveyed long-term protection to mice against an i.n. challenge with heterologous HSV-2. We now show that i.n. immunization of female mice with this same vector, AdgB8, provides secretory and serum-derived humoral immune responses in the genital tract. Intranasal immunization induced anti-HSVgB IgA and IgG in vaginal washes of mice, whereas i.p. immunization only induced IgG, which appeared to be serum-derived. Interestingly, intravaginal (ivag) immunization with AdgB8 resulted in little or no anti-HSVgB IgA and only low levels of specific IgG in vaginal washes. All three routes of inoculation induced gB-specific serum IgG and IgA; however, i.n. immunized mice demonstrated the highest level of serum anti-HSVgB IgA. Additionally, ivag boosting with AdgB8 did not significantly alter the serum or vaginal wash antibody responses in i.n. or i.p. immunized mice. The IgG to IgA ratios of gB-specific and total antibody titres in the serum and vaginal washes of i.n. immunized mice indicated that the IgA in the vaginal washes was likely to be secretory. Furthermore, the titres of anti-HSVgB IgA relative to total IgA were higher in vaginal washes than sera, suggesting that the gB-specific vaginal wash IgA present in i.n. immunized mice was locally produced.

Use of human adenovirus-based vectors for antigen expression in animals.

An infectious recombinant human adenovirus type 5 (Ad5) vector, AdG12, which carries the glycoprotein gene of vesicular stomatitis virus (VSV) and expresses that gene in cultured HeLa cells was used to examine the host range of insert expression by human Ad vectors. The VSV glycoprotein was expressed in bovine, canine and murine cells when infected with AdG12 in culture. These cell lines are respectively permissive, non-permissive and semi-permissive for human Ad5 replication. Administration of the AdG12 vector to calves, piglets or dogs by either the subcutaneous or oral route resulted in the production of high titres of neutralizing antibodies to VSV. Mice injected intraperitoneally with the vector produced neutralizing antibodies and were protected against subsequent intravenous challenge with normally lethal doses of VSV. This work demonstrates the utility of human adenoviral vectors for antigen expression in a number of non-human cell lines and for the induction of an immune response to the delivered antigen in a number of species.

Toll-Like Receptor (TLR)-3, but Not TLR4, Agonist Protects against Genital Herpes Infection in the Absence of Inflammation Seen with CpG DNA

We previously demonstrated that delivery of CpG oligodeoxynucleotide (ODN) to vaginal mucosa induced an innate mucosal antiviral state that protected against intravaginal challenge with herpes simplex virus (HSV)-2. We report that mucosal, but not systemic, delivery of ligands for Toll-like receptor (TLR)-3, but not TLR4, induced protection against genital HSV-2 challenge that was not accompanied by the local inflammation and splenomegaly seen after treatment with CpG ODN. Surprisingly, TLR4 messenger (m) RNA expression was shown to be higher than that of TLR3 or TLR9 in murine genital mucosa. Similarly, murine RAW264.7 cells were shown to express more mRNA for TLR4 than TLR3 or TLR9, yet treatment of these cells with double-stranded RNA provided greater protection than lipopolysaccharide or CpG ODN. These results indicate that TLR3 ligand induces a more potent antiviral response than TLR4 and TLR9 ligands and may be a safer means of protecting against sexually transmitted viral infections.

Long-Term Immunity and Protection against Herpes Simplex Virus Type 2 in the Murine Female Genital Tract after Mucosal but Not Systemic Immunization

The degree and duration of immunity against herpes simplex virus type 2 (HSV-2) infection of the female genital tract were assessed after intranasal (i.nl.) or intraperitoneal (i.p.) immunization with a recombinant adenovirus vector expressing HSV glycoprotein B (AdgB8). After intravaginal HSV-2 challenge, control mice rapidly developed disease and displayed high virus titers in vaginal washes. In contrast, virus titers decreased significantly and at similar rates in i.nl. and i.p. immunized mice and by day 7 were undetectable in vaginal wash samples. Assessment of genital pathology and survival showed that only i.nl. immunization provided long-term protection. Examination of antibody-secreting cells (ASCs) during the decline in vaginal virus titers revealed that gB-specific IgA ASCs were only observed in the genital tissues of i.nl. immunized mice. These results indicate that mucosal immunization provides a high and long-lasting level of immunity from sexually transmitted viral infections of the female genital tract.

Toll-like Receptor 9, CpG DNA and Innate Immunity

Innate immunity provides the first line of defense against invading pathogens and is essential for survival in the absence of adaptive immune responses. Innate immune recognition relies on a limited number of germ-line encoded receptors, such as Toll-like receptors (TLRs), that evolved to recognize conserved molecular patterns of microbial origin. To date, ten transmembrane proteins in the TLR family have been described. It is becoming increasingly clear that bacterial CpG DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG are potent inducers of the innate immune system including dendritic cells (DCs), macrophages, and natural killer (NK) and NKT cells. Recent studies indicate that mucosal or systemic delivery of CpG DNA can act as a potent adjuvant in a vaccine combination or act alone as an anti-microbial agent. Recently, it was shown that TLR9 is essential for the recognition of unmethylated CpG DNA since cells from TLR9-deficient mice are unresponsive to CpG stimulation. Although the effects of CpG DNA on bone marrow-derived cells are beginning to unfold, there has been little or no information regarding the mechanisms of CpG DNA function on non-immune cells or tissues. This review focuses on the recent advances in CpG-DNA/TLR9 signaling effects on the activation of innate immunity.

Toll-like receptor expression and responsiveness are increased in viraemic HIV-1 infection

Objectives:Toll-like receptors (TLR) are important in pathogen recognition and may play a role in HIV disease. We evaluated the effect of chronic untreated and treated HIV-1 infection on systemic TLR expression and TLR signalling. Methods:Two hundred HIV-infected and uninfected women from a Kenya cohort participated in the studies. TLR1 to TLR10 messenger RNA expression was determined by quantitative reverse transcriptase polymerase chain reaction in peripheral blood mononuclear cells (PBMC). TLR ligand responsiveness was determined in or using ex-vivo PBMC by cytokine production in culture supernatants. Results:Chronic, untreated HIV-1 infection was significantly associated with increased mRNA expression of TLR6, TLR7, and TLR8 and when analysis was limited to those with advanced disease (CD4 cell count < 200 cells/ml) TLR2, TLR3, and TLR4 were additionally elevated. TLR expression correlated with the plasma HIV-RNA load, which was significant for TLR6 and TLR7. In vitro HIV single-stranded RNA alone could enhance TLR mRNA expression. PBMC of HIV-infected subjects also demonstrated profoundly increased proinflammatory responsiveness to TLR ligands, suggesting sensitization of TLR signalling in HIV. Finally, viral suppression by HAART was associated with a normalization of TLR levels. Conclusion:Together, these data indicate that chronic viraemic HIV-1 is associated with increased TLR expression and responsiveness, which may perpetuate innate immune dysfunction and activation that underlies HIV pathogenesis, and thus reveal potential new targets for therapy.