Breadth and Functionality of Varicella-Zoster Virus Glycoprotein-Specific Antibodies Identified after Zostavax Vaccination in Humans.

Herpes zoster (HZ) (shingles) is the clinical manifestation of varicella-zoster virus (VZV) reactivation. HZ typically develops as people age, due to decreased cell-mediated immunity. However, the importance of antibodies for immunity against HZ prevention remains to be understood. The goal of this study was to examine the breadth and functionality of VZV-specific antibodies after vaccination with a live attenuated HZ vaccine (Zostavax). Direct enumeration of VZV-specific antibody-secreting cells (ASCs) via enzyme-linked immunosorbent spot assay (ELISPOT assay) showed that Zostavax can induce both IgG and IgA ASCs 7 days after vaccination but not IgM ASCs. The VZV-specific ASCs range from 33 to 55% of the total IgG ASCs. Twenty-five human VZV-specific monoclonal antibodies (MAbs) were cloned and characterized from single-cell-sorted ASCs of five subjects (>60 years old) who received Zostavax. These MAbs had an average of ∼20 somatic hypermutations per VH gene, similar to those seen after seasonal influenza vaccination. Fifteen of the 25 MAbs were gE specific, whereas the remaining MAbs were gB, gH, or gI specific. The most potent neutralizing antibodies were gH specific and were also able to inhibit cell-to-cell spread of the virus in vitro Most gE-specific MAbs were able to neutralize VZV, but they required the presence of complement and were unable to block cell-to-cell spread. These data indicate that Zostavax induces a memory B cell recall response characterized by anti-gE > anti-gI > anti-gB > anti-gH antibodies. While antibodies to gH could be involved in limiting the spread of VZV upon reactivation, the contribution of anti-gE antibodies toward protective immunity after Zostavax needs further evaluation.IMPORTANCE Varicella-zoster virus (VZV) is the causative agent of chickenpox and shingles. Following infection with VZV, the virus becomes latent and resides in nerve cells. Age-related declines in immunity/immunosuppression can result in reactivation of this latent virus, causing shingles. It has been shown that waning T cell immunity correlates with an increased incidence of VZV reactivation. Interestingly, serum with high levels of VZV-specific antibodies (VariZIG; IV immunoglobulin) has been administered to high-risk populations, e.g., immunocompromised children, newborns, and pregnant women, after exposure to VZV and has shown some protection against chickenpox. However, the relative contribution of antibodies against individual surface glycoproteins toward protection from shingles in elderly/immunocompromised individuals has not been established. Here, we examined the breadth and functionality of VZV-specific antibodies after vaccination with the live attenuated VZV vaccine Zostavax in humans. This study will add to our understanding of the role of antibodies in protection against shingles.

Effector and Central Memory Poly-Functional CD4(+) and CD8(+) T Cells are Boosted upon ZOSTAVAX(®) Vaccination.

ZOSTAVAX(®) is a live attenuated varicella-zoster virus (VZV) vaccine that is licensed for the protection of individuals ≥50 years against shingles and its most common complication, postherpetic neuralgia. While IFNγ responses increase upon vaccination, the quality of the T cell response has not been elucidated. By using polychromatic flow cytometry, we characterized the breadth, magnitude, and quality of ex vivo CD4(+) and CD8(+) T cell responses induced 3-4 weeks after ZOSTAVAX vaccination of healthy adults. We show, for the first time that the highest frequencies of VZV-specific CD4(+) T cells were poly-functional CD154(+)IFNγ(+)IL-2(+)TNFα(+) cells, which were boosted upon vaccination. The CD4(+) T cells were broadly reactive to several VZV proteins, with immediate early (IE) 63 ranking the highest among them in the fold rise of poly-functional cells, followed by IE62, gB, open reading frame (ORF) 9, and gE. We identified a novel poly-functional ORF9-specific CD8(+) T cell population in 62% of the subjects, and these were boosted upon vaccination. Poly-functional CD4(+) and CD8(+) T cells produced significantly higher levels of IFNγ, IL-2, and TNFα compared to mono-functional cells. After vaccination, a boost in the expression of IFNγ by poly-functional IE63- and ORF9-specific CD4(+) T cells and IFNγ, IL-2, and TNFα by ORF9-specific poly-functional CD8(+) T cells was observed. Responding poly-functional T cells exhibited both effector (CCR7(-)CD45RA(-)CD45RO(+)), and central (CCR7(+)CD45RA(-)CD45RO(+)) memory phenotypes, which expressed comparable levels of cytokines. Altogether, our studies demonstrate that a boost in memory poly-functional CD4(+) T cells and ORF9-specific CD8(+) T cells may contribute toward ZOSTAVAX efficacy.

Abortive intrabronchial infection of rhesus macaques with varicella-zoster virus provides partial protection against simian varicella virus challenge.

UNLABELLED: Varicella-zoster virus (VZV) is a human neurotropic alphaherpesvirus and the etiological agent of varicella (chickenpox) and herpes zoster (HZ, shingles). Previously, inoculation of monkeys via the subcutaneous, intratracheal, intravenous, or oral-nasal-conjunctival routes did not recapitulate all the hallmarks of VZV infection, including varicella, immunity, latency, and reactivation. Intrabronchial inoculation of rhesus macaques (RMs) with simian varicella virus (SVV), a homolog of VZV, recapitulates virologic and immunologic hallmarks of VZV infection in humans. Given that VZV is acquired primarily via the respiratory route, we investigated whether intrabronchial inoculation of RMs with VZV would result in a robust model. Despite the lack of varicella and viral replication in either the lungs or whole blood, all four RMs generated an immune response characterized by the generation of VZV-specific antibodies and T cells. Two of 4 VZV-inoculated RMs were challenged with SVV to determine cross-protection. VZV-immune RMs displayed no varicella rash and had lower SVV viral loads and earlier and stronger humoral and cellular immune responses than controls. In contrast to the results for SVV DNA, no VZV DNA was detected in sensory ganglia at necropsy. In summary, following an abortive VZV infection, RMs developed an adaptive immune response that conferred partial protection against SVV challenge. These data suggest that a replication-incompetent VZV vaccine that does not establish latency may provide sufficient protection against VZV disease and that VZV vaccination of RMs followed by SVV challenge provides a model to evaluate new vaccines and therapeutics against VZV. IMPORTANCE: Although VZV vaccine strain Oka is attenuated, it can cause mild varicella, establish latency, and in rare cases, reactivate to cause herpes zoster (HZ). Moreover, studies suggest that the HZ vaccine (Zostavax) only confers short-lived immunity. The development of more efficacious vaccines would be facilitated by a robust animal model of VZV infection. The data presented in this report show that intrabronchial inoculation of rhesus macaques (RMs) with VZV resulted in an abortive VZV infection. Nevertheless, all animals generated a humoral and cellular immune response that conferred partial cross-protection against simian varicella virus (SVV) challenge. Additionally, VZV DNA was not detected in the sensory ganglia, suggesting that viremia might be required for the establishment of latency. Therefore, VZV vaccination of RMs followed by SVV challenge is a model that will support the development of vaccines that boost protective T cell responses against VZV.

Human sensory neurons derived from induced pluripotent stem cells support varicella-zoster virus infection.

After primary infection, varicella-zoster virus (VZV) establishes latency in neurons of the dorsal root and trigeminal ganglia. Many questions concerning the mechanism of VZV pathogenesis remain unanswered, due in part to the strict host tropism and inconsistent availability of human tissue obtained from autopsies and abortions. The recent development of induced pluripotent stem (iPS) cells provides great potential for the study of many diseases. We previously generated human iPS cells from skin fibroblasts by introducing four reprogramming genes with non-integrating adenovirus. In this study, we developed a novel protocol to generate sensory neurons from iPS cells. Human iPS cells were exposed to small molecule inhibitors for 10 days, which efficiently converted pluripotent cells into neural progenitor cells (NPCs). The NPCs were then exposed for two weeks to growth factors required for their conversion to sensory neurons. The iPS cell-derived sensory neurons were characterized by immunocytochemistry, flow cytometry, RT-qPCR, and electrophysiology. After differentiation, approximately 80% of the total cell population expressed the neuron-specific protein, ßIII-tubulin. Importantly, 15% of the total cell population co-expressed the markers Brn3a and peripherin, indicating that these cells are sensory neurons. These sensory neurons could be infected by both VZV and herpes simplex virus (HSV), a related alphaherpesvirus. Since limited neuronal populations are capable of supporting the entire VZV and HSV life cycles, our iPS-derived sensory neuron model may prove useful for studying alphaherpesvirus latency and reactivation.

Varicella zoster virus induces neuropathic changes in rat dorsal root ganglia and behavioral reflex sensitisation that is attenuated by gabapentin or sodium channel blocking drugs.

Reactivation of latent varicella zoster virus (VZV) within sensory trigeminal and dorsal root ganglia (DRG) neurons produces shingles (zoster), often accompanied by a chronic neuropathic pain state, post-herpetic neuralgia (PHN). PHN persists despite latency of the virus within human sensory ganglia and is often unresponsive to current analgesic or antiviral agents. To study the basis of varicella zoster-induced pain, we have utilised a recently developed model of chronic VZV infection in rodents. Immunohistochemical analysis of DRG following VZV infection showed the presence of a viral immediate early gene protein (IE62) co-expressed with markers of A- (neurofilament-200; NF-200) and C- (peripherin) afferent sensory neurons. There was increased expression of neuropeptide Y (NPY) in neurons co-expressing NF-200. In addition, there was an increased expression of alpha2delta1 calcium channel, Na(v)1.3 and Na(v)1.8 sodium channels, the neuropeptide galanin and the nerve injury marker, Activating Transcription Factor-3 (ATF-3) as determined by Western blotting in DRG of VZV-infected rats. VZV infection induced increased behavioral reflex responsiveness to both noxious thermal and mechanical stimuli ipsilateral to injection (lasting up to 10 weeks post-infection) that is mediated by spinal NMDA receptors. These changes were reversed by systemic administration of gabapentin or the sodium channel blockers, mexiletine and lamotrigine, but not by the non-steroidal anti-inflammatory agent, diclofenac. This is the first time that the profile of VZV infection-induced phenotypic changes in DRG has been shown in rodents and reveals that this profile appears to be broadly similar (but not identical) to changes in other neuropathic pain models.

Memory T-cell response to rotavirus detected with a gamma interferon enzyme-linked immunospot assay.

Measurements of serum-neutralizing antibody and anti-rotavirus immunoglobulin A (IgA) are the current standard for assessing immune responses following rotavirus vaccination. However, there is ongoing debate as to whether antibody titers correlate with protection against rotavirus gastroenteritis. Children recovering from rotavirus gastroenteritis have increased gamma interferon release from cultured peripheral blood mononuclear cells (PBMCs), suggesting that cell-mediated immunity (CMI) may play a role in viral clearance and protection from subsequent gastroenteritis. We have developed a gamma interferon enzyme-linked immunospot (ELISPOT) assay for evaluation of CMI responses to rotavirus using frozen PBMCs obtained from healthy adults. Responses to three different rotavirus antigen types were analyzed-a peptide pool based on the human VP6 sequence; reassortant human:bovine vaccine strains; and cell culture-adapted (CCA) human G1, G2, G3, G4, and bovine (WC3) G6 strains. The reassortant strains consist of a bovine WC3 genome background expressing the human rotavirus surface proteins VP7 (G1, G2, G3, or G4) or VP4 (P1). Responses to titrations of the peptide pool as well as CCA and reassortant strains were assessed. Gamma interferon ELISPOT responses were similar for CCA and reassortant strains, whether live or UV inactivated, and when tested either individually or pooled. For most subjects, responses to the VP6 peptide pool positively correlated with responses to CCA and reassortant strains. Cell depletion studies indicate the memory responses detected with these frozen adult PBMCs were primarily due to the CD4+ T-cell population. This gamma interferon ELISPOT assay provides a new tool to apply in clinical studies for the characterization of natural or vaccine-induced CMI to rotavirus.

Safety, efficacy, and immunogenicity of a live, quadrivalent human-bovine reassortant rotavirus vaccine in healthy infants.

OBJECTIVES: To investigate safety, efficacy, and immunogenicity of live quadrivalent rotavirus vaccine (QRV) containing human-bovine (WC3) reassortant rotavirus serotypes G1, G2, G3, and P1a. STUDY DESIGN: This was a randomized, double-blinded, placebo-controlled trial. During 1993 to 1994, at 10 US study sites, 439 healthy infants approximately 2 to 6 months of age, were enrolled to receive 3 doses of oral QRV or placebo at approximately 8-week intervals. RESULTS: The vaccine was generally well tolerated; no serious vaccine-related adverse experiences were reported. Risk differences and 95% confidence intervals suggested no differences between vaccine and placebo recipients in the incidences of fever, irritability, vomiting, or diarrhea during the 14 days after any dose. QRV was 74.6% efficacious (95% CI: 49.5%, 88.3%) in preventing rotavirus acute gastroenteritis (AGE), regardless of severity and 100% efficacious (95% CI: 43.5%, 100%) in preventing severe rotavirus AGE through one rotavirus season. Serotype G1 was identified in most infants with rotavirus AGE. A >or=3-fold rise in serum neutralizing antibody to G1 was observed in 57% (45/79) of vaccinees. A >or=3-fold rise in serum anti-rotavirus IgA and fecal anti-rotavirus IgA was observed in 88% (162/185) and 65% (104/159) of vaccinees, respectively. CONCLUSIONS: QRV was generally well tolerated, immungenic, and highly effective against rotavirus gastroenteritis.

Safety, immunogenicity and efficacy in healthy infants of G1 and G2 human reassortant rotavirus vaccine in a new stabilizer/buffer liquid formulation.

BACKGROUND: A refrigerator-stable rotavirus (RV) vaccine that withstands gastric acid is anticipated to permit more widespread use of RV vaccine. OBJECTIVE: We investigated for the first time in infants an oral, liquid formulation of G1 and G2 human bovine reassortant rotavirus vaccine (HRRV) with a new stabilizer/buffer (S/B) containing sucrose, sodium phosphate and sodium citrate. METHODS: During 1997 through 1998, 731 healthy infants approximately 2 to 4 months of age were enrolled at 19 US sites to receive 3 HRRV or placebo doses approximately 6 to 8 weeks apart in a partially double blinded study. Infants were randomized to: (1) HRRV with no S/B but with prefeeding; (2) HRRV plus 1 of 3 different concentrations/volumes of S/B; or (3) placebo. RESULTS: No serious vaccine-related adverse experiences or intussusception cases were reported. No statistically significant differences were observed between vaccine and placebo recipients for fever (> or =38.1 degrees C) 0 to 7 days after any dose, irritability, vomiting or diarrhea incidence 0 to 42 days after any dose. Vaccine virus shedding among vaccine recipients was uncommon. Among S/B vaccine groups, proportions of infants with a > or =3-fold titer rise from baseline to Postdose 3 for G1 serum-neutralizing antibody (SNA), G2 SNA, WC3 SNA, serum anti-RV IgA, serum anti-RV IgG and stool anti-RV IgA were generally similar to those of the prefed non-S/B group. CONCLUSIONS: HRRV with a new S/B was generally well-tolerated; immunogenicity was generally similar to the prefed non-S/B group. No intussusception cases were reported, but the small sample size precluded a definitive conclusion. A large international clinical study is under way to address safety and efficacy of an S/B formulation of a pentavalent version of HRRV.