Kaposi sarcoma-associated herpesvirus complement control protein (KCP) and glycoprotein K8.1 are not required for viral infection in vitro or in vivo.

Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causal agent of Kaposi sarcoma, a cancer that appears as tumors on the skin or mucosal surfaces, as well as primary effusion lymphoma and KSHV-associated multicentric Castleman disease, which are B-cell lymphoproliferative disorders. Effective prophylactic and therapeutic strategies against KSHV infection and its associated diseases are needed. To develop these strategies, it is crucial to identify and target viral glycoproteins involved in KSHV infection of host cells. Multiple KSHV glycoproteins expressed on the viral envelope are thought to play a pivotal role in viral infection, but the infection mechanisms involving these glycoproteins remain largely unknown. We investigated the role of two KSHV envelope glycoproteins, KSHV complement control protein (KCP) and K8.1, in viral infection in various cell types in vitro and in vivo. Using our newly generated anti-KCP antibodies, previously characterized anti-K8.1 antibodies, and recombinant mutant KSHV viruses lacking KCP, K8.1, or both, we demonstrated the presence of KCP and K8.1 on the surface of both virions and KSHV-infected cells. We showed that KSHV lacking KCP and/or K8.1 remained infectious in KSHV-susceptible cell lines, including epithelial, endothelial, and fibroblast, when compared to wild-type recombinant KSHV. We also provide the first evidence that KSHV lacking K8.1 or both KCP and K8.1 can infect human B cells in vivo in a humanized mouse model. Thus, these results suggest that neither KCP nor K8.1 is required for KSHV infection of various host cell types and that these glycoproteins do not determine KSHV cell tropism. IMPORTANCE: Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic human gamma-herpesvirus associated with the endothelial malignancy Kaposi sarcoma and the lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman disease. Determining how KSHV glycoproteins such as complement control protein (KCP) and K8.1 contribute to the establishment, persistence, and transmission of viral infection will be key for developing effective anti-viral vaccines and therapies to prevent and treat KSHV infection and KSHV-associated diseases. Using newly generated anti-KCP antibodies, previously characterized anti-K8.1 antibodies, and recombinant mutant KSHV viruses lacking KCP and/or K8.1, we show that KCP and K8.1 can be found on the surface of both virions and KSHV-infected cells. Furthermore, we show that KSHV lacking KCP and/or K8.1 remains infectious to diverse cell types susceptible to KSHV in vitro and to human B cells in vivo in a humanized mouse model, thus providing evidence that these viral glycoproteins are not required for KSHV infection.

Kaposi Sarcoma-Associated Herpesvirus Glycoprotein H Is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types.

Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and is the causative infectious agent of Kaposi sarcoma and two malignancies of B cell origin. To date, there is no licensed KSHV vaccine. Development of an effective vaccine against KSHV continues to be limited by a poor understanding of how the virus initiates acute primary infection in vivo in diverse human cell types. The role of glycoprotein H (gH) in herpesvirus entry mechanisms remains largely unresolved. To characterize the requirement for KSHV gH in the viral life cycle and in determination of cell tropism, we generated and characterized a mutant KSHV in which expression of gH was abrogated. Using a bacterial artificial chromosome containing a complete recombinant KSHV genome and recombinant DNA technology, we inserted stop codons into the gH coding region. We used electron microscopy to reveal that the gH-null mutant virus assembled and exited from cells normally, compared to wild-type virus. Using purified virions, we assessed infectivity of the gH-null mutant in diverse mammalian cell types in vitro Unlike wild-type virus or a gH-containing revertant, the gH-null mutant was unable to infect any of the epithelial, endothelial, or fibroblast cell types tested. However, its ability to infect B cells was equivocal and remains to be investigated in vivo due to generally poor infectivity in vitro Together, these results suggest that gH is critical for KSHV infection of highly permissive cell types, including epithelial, endothelial, and fibroblast cells.IMPORTANCE All homologues of herpesvirus gH studied to date have been implicated in playing an essential role in viral infection of diverse permissive cell types. However, the role of gH in the mechanism of KSHV infection remains largely unresolved. In this study, we generated a gH-null mutant KSHV and provided evidence that deficiency of gH expression did not affect viral particle assembly or egress. Using the gH-null mutant, we showed that gH was indispensable for KSHV infection of epithelial, endothelial, and fibroblast cells in vitro This suggests that gH is an important target for the development of a KSHV prophylactic vaccine to prevent initial viral infection.

High Epstein-Barr Virus Load and Genomic Diversity Are Associated with Generation of gp350-Specific Neutralizing Antibodies following Acute Infectious Mononucleosis.

The Epstein-Barr virus (EBV) gp350 glycoprotein interacts with the cellular receptor to mediate viral entry and is thought to be the major target for neutralizing antibodies. To better understand the role of EBV-specific antibodies in the control of viral replication and the evolution of sequence diversity, we measured EBV gp350-specific antibody responses and sequenced the gp350 gene in samples obtained from individuals experiencing primary EBV infection (acute infectious mononucleosis [AIM]) and again 6 months later (during convalescence [CONV]). EBV gp350-specific IgG was detected in the sera of 17 (71%) of 24 individuals at the time of AIM and all 24 (100%) individuals during CONV; binding antibody titers increased from AIM through CONV, reaching levels equivalent to those in age-matched, chronically infected individuals. Antibody-dependent cell-mediated phagocytosis (ADCP) was rarely detected during AIM (4 of 24 individuals; 17%) but was commonly detected during CONV (19 of 24 individuals; 79%). The majority (83%) of samples taken during AIM neutralized infection of primary B cells; all samples obtained at 6 months postdiagnosis neutralized EBV infection of cultured and primary target cells. Deep sequencing revealed interpatient gp350 sequence variation but conservation of the CR2-binding site. The levels of gp350-specific neutralizing activity directly correlated with higher peripheral blood EBV DNA levels during AIM and a greater evolution of diversity in gp350 nucleotide sequences from AIM to CONV. In summary, we conclude that the viral load and EBV gp350 diversity during early infection are associated with the development of neutralizing antibody responses following AIM. IMPORTANCE: Antibodies against viral surface proteins can blunt the spread of viral infection by coating viral particles, mediating uptake by immune cells, or blocking interaction with host cell receptors, making them a desirable component of a sterilizing vaccine. The EBV surface protein gp350 is a major target for antibodies. We report the detection of EBV gp350-specific antibodies capable of neutralizing EBV infection in vitro The majority of gp350-directed vaccines focus on glycoproteins from lab-adapted strains, which may poorly reflect primary viral envelope diversity. We report some of the first primary gp350 sequences, noting that the gp350 host receptor binding site is remarkably stable across patients and time. However, changes in overall gene diversity were detectable during infection. Patients with higher peripheral blood viral loads in primary infection and greater changes in viral diversity generated more efficient antibodies. Our findings provide insight into the generation of functional antibodies, necessary for vaccine development.

Screening for cervical cancer among HIV-positive and HIV-negative women in Cameroon using simultaneous co-testing with careHPV DNA testing and visual inspection enhanced by digital cervicography: Findings of initial screening and one-year follow-up.

OBJECTIVE: The World Health Organization (WHO)'s cervical cancer screening guidelines for limited-resource settings recommend sequential screening followed by same-day treatment under a "screen-and-treat" approach. We aimed to (1) assess feasibility and clinical outcomes of screening HIV-positive and HIV-negative Cameroonian women by pairing visual inspection with acetic acid and Lugol's iodine enhanced by digital cervicography (VIA/VILI-DC) with careHPV, a high-risk human papillomavirus (HR-HPV) nucleic acid test designed for low-resource settings; and (2) determine persistence of HR-HPV infection after one-year follow-up to inform optimal screening, treatment, and follow-up algorithms. METHODS: We co-tested 913 previously unscreened women aged ≥30years and applied WHO-recommended treatment for all VIA/VILI-DC-positive women. Baseline prevalence of HR-HPV and HIV were 24% and 42%, respectively. RESULTS: On initial screen, 44 (5%) women were VIA/VILI-DC-positive, of whom 22 had HR-HPV infection, indicating 50% of women screened false-positive and would have been triaged for unnecessary same-day treatment. VIA/VILI-DC-positive women with HIV infection were three times more likely to be HR-HPV-positive than HIV-negative women (65% vs. 20%). All women positive for either VIA/VILI-DC or HR-HPV (n=245) were invited for repeat co-testing after one year, of which 136 (56%) returned for follow-up. Of 122 women who were HR-HPV-positive on initial screen, 60 (49%) re-tested negative, of whom 6 had received treatment after initial screen, indicating that 44% of initially HR-HPV-positive women spontaneously cleared infection after one year without treatment. Women with HIV were more likely to remain HR-HPV-positive on follow-up than HIV-negative women (61% vs. 22%, p<0.001). Treatment was offered to all VIA/VILI-DC positive women on initial screen, and to all women screening VIA/VILI-DC or HR-HPV positive on follow-up. CONCLUSIONS: We found careHPV co-testing with VIA/VILI-DC to be feasible and valuable in identifying false-positives, but careHPV screening-to-result time was too long to inform same-day treatment.

Implementing a Fee-for-Service Cervical Cancer Screening and Treatment Program in Cameroon: Challenges and Opportunities.

BACKGROUND: Cervical cancer screening is one of the most effective cancer prevention strategies, but most women in Africa have never been screened. In 2007, the Cameroon Baptist Convention Health Services, a large faith-based health care system in Cameroon, initiated the Women's Health Program (WHP) to address this disparity. The WHP provides fee-for-service cervical cancer screening using visual inspection with acetic acid enhanced by digital cervicography (VIA-DC), prioritizing care for women living with HIV/AIDS. They also provide clinical breast examination, family planning (FP) services, and treatment for reproductive tract infection (RTI). Here, we document the strengths and challenges of the WHP screening program and the unique aspects of the WHP model, including a fee-for-service payment system and the provision of other women's health services. METHODS: We retrospectively reviewed WHP medical records from women who presented for cervical cancer screening from 2007-2014. RESULTS: In 8 years, WHP nurses screened 44,979 women for cervical cancer. The number of women screened increased nearly every year. The WHP is sustained primarily on fees-for-service, with external funding totaling about $20,000 annually. In 2014, of 12,191 women screened for cervical cancer, 99% received clinical breast exams, 19% received FP services, and 4.7% received treatment for RTIs. We document successes, challenges, solutions implemented, and recommendations for optimizing this screening model. CONCLUSION: The WHP's experience using a fee-for-service model for cervical cancer screening demonstrates that in Cameroon VIA-DC is acceptable, feasible, and scalable and can be nearly self-sustaining. Integrating other women's health services enabled women to address additional health care needs. IMPLICATION FOR PRACTICE: The Cameroon Baptist Convention Health Services Women's Health Program successfully implemented a nurse-led, fee-for-service cervical cancer screening program using visual inspection with acetic acid-enhanced by digital cervicography in the setting of a large faith-based health care system in Cameroon. It is potentially replicable in many African countries, where faith-based organizations provide a large portion of health care. The cost-recovery model and concept of offering multiple services in a single clinic rather than stand-alone "silo" cervical cancer screening could provide a model for other low-and-middle-income countries planning to roll out a new, or make an existing, cervical cancer screening services accessible, comprehensive, and sustainable.

A chimeric EBV gp350/220-based VLP replicates the virion B-cell attachment mechanism and elicits long-lasting neutralizing antibodies in mice.

Epstein-Barr virus (EBV), an oncogenic gammaherpesvirus, causes acute infectious mononucleosis (AIM) and is linked to the development of several human malignancies. There is an urgent need for a vaccine that is safe, prevents infection and/or limits disease. Unique among human herpesviruses, glycoprotein (gp)350/220, which initiates EBV attachment to susceptible host cells, is the major ligand on the EBV envelope and is highly conserved. Interaction between gp350/220 and complement receptor type 2 (CR2)/CD21 and/or (CR1)/CD35 on B-cells is required for infection. Potent antibody responses to gp350/220 occur in animal models and humans. Thus, gp350/220 provides an attractive candidate for prophylactic subunit vaccine development. However, in a recent Phase II clinical trial immunization with soluble recombinant gp350 reduced the incidence of AIM, but did not prevent infection. Despite various attempts to produce an EBV vaccine, no vaccine is licensed. Herein we describe a sub-unit vaccine against EBV based on a novel Newcastle disease virus (NDV)-virus-like particle (VLP) platform consisting of EBVgp350/220 ectodomain fused to NDV-fusion (F) protein. The chimeric protein EBVgp350/220-F is incorporated into the membrane of a VLP composed of the NDV matrix and nucleoprotein. The particles resemble native EBV in diameter and shape and bind CD21 and CD35. Immunization of BALB/c mice with EBVgp350/220-F VLPs elicited strong, long-lasting neutralizing antibody responses when assessed in vitro. This chimeric VLP is predicted to provide a superior safety profile as it is efficiently produced in Chinese hamster ovary (CHO) cells using a platform devoid of human nucleic acid and EBV-transforming genes.

Cervical Cancer Screening in Cameroon: Interobserver Agreement on the Interpretation of Digital Cervicography Results.

OBJECTIVE: The World Health Organization recommends visual inspection with acetic acid (VIA) for cervical cancer screening in resource-limited settings. In Cameroon, we use digital cervicography (DC) to capture images of the cervix after VIA. This study evaluated interobserver agreement of DC results, compared DC with histopathologic results, and examined interobserver agreement among screening methods. METHOD: Three observers, blinded to each other's interpretations, evaluated 540 DC photographs as follows: (1) negative/positive for acetowhite lesions or cancer and (2) assigned a presumptive diagnosis of histopathologic lesion grade in the 91 cases that had a histopathologic diagnosis. Observer A was the actual screening nurse; B, a reproductive health nurse; C, a gynecologic oncologist; and D, the histopathologic diagnosis. We compared inter-rater agreement of DC impressions among observers A, B, and C, and with D, with Cohen kappas. RESULTS: For interpretations of DC, (negative/positive) strengths of agreement of paired observers were the following: A/B, moderate [K, 0.54; 95% confidence interval (CI), 0.47-0.61], A/C, fair (K, 0.37; 95% CI, 0.29-0.44), and B/C, moderate (K, 0.45; 95% CI, 0.37-0.53). For presumptive pathologic grading, strengths of agreement for weighted Ks were as follows: A/B, moderate (K, 0.42; 95% CI, 0.28-0.56); A/C, fair (K, 0.33; 95% CI, 0.20-0.46); B/C, fair (K, 0.54; 95% CI, 0.40-0.67); A/D, moderate (K, 0.59; 95% CI, 0.45-0.74); B/D, moderate (K, 0.58; 95% CI, 0.46-0.70); and C/D, moderate (K, 0.50; 95% CI, 0.37-0.63). CONCLUSIONS: Interobserver agreement of DC interpretations was mostly moderate among the 3 observers, between them and histopathology, and comparable to that of other visual-based screening methods, i.e., VIA, cytology, or colposcopy.

SIRPα/CD172a and FHOD1 are unique markers of littoral cells, a recently evolved major cell population of red pulp of human spleen.

Asplenic individuals are compromised not only in their ability to destroy infectious agents, but are at increased risk for death from autoimmune disease, certain tumors, and ischemic heart disease. Enhanced mortality is attributed to lack of phagocytes sequestered in spleen that efficiently engulf and destroy appropriate targets, although related cells are found elsewhere. To determine whether a unique population regulates RBC-pathogen clearance and filtration of altered self, we reviewed the anatomic literature and analyzed in situ by immunohistochemistry and immunofluorescence the expression patterns of a little-characterized cell that dominates the splenic red pulp of humans and closely related primates: the venous sinus-lining or littoral cell (LC). High expression of the formin homology domain protein 1 outlines the LC population. Although LCs are endothelial-like in distribution, they express several macrophage-directed proteins, the RBC Duffy Ag receptor for chemokines and T cell coreceptor CD8α/α, yet they lack lineage-associated markers CD34 and CD45. Strikingly, SIRPα (CD172a) expression in human spleen concentrates on LCs, consistent with recent demonstration of a key role in RBC turnover and elimination versus release of infected or altered self. Our results indicate human LCs (SIRPα(+), formin homology domain protein 1(+), CD8α/α(+), CD34(-), CD45(-)) comprise a highly plastic barrier cell population that emerged late in primate evolution coordinate with CD8 expression. Unique to Hominidae, LCs may be the ultimate determinant of which cells recirculate after passage through human spleen.

Awareness, acceptability and uptake of human papilloma virus vaccine among Cameroonian school-attending female adolescents.

The purpose of this study is to assess awareness, knowledge and beliefs about HPV, cervical cancer HPV vaccine and willingness to vaccinate among adolescent females aged 12-26 years at clinics and schools in the North West Region of Cameroon where the Cameroon Baptist Convention Health Services (CBCHS) conducted a sensitization and education campaign. A questionnaire survey was administered among female adolescents in schools and clinics. Descriptive statistics including frequencies, percentages and proportions were generated for independent variables and bivariate analyses (Chi square test) were used to assess the relationship between independent and outcome variables. Data were analyzed using SAS Version 9.2. Out of a sample of 650 adolescent girls 553 willingly participated in this anonymous survey. Awareness of HPV (86.8%), cervical cancer (82.3%), and prevention of HPV infections through vaccination (75.9%) was significantly higher than in previous studies conducted in Cameroon and other developing countries. Higher perceptions of risk were associated with being sexually active (p = 0.0013), age (p = 0.0031) and level of education (p = 0.0274). Age and educational level were associated with HPV knowledge (p = 0.007 and p = 0.0008), respectively. The mean level of interest in HPV vaccination was 4.0 (SD = 1.27), which is above the mean. Our data indicate high awareness about HPV, cervical cancer and HPV vaccine among adolescents, and interest in receiving the vaccine and learning about the disease. CBCHS' community education strategy effectively raised acceptability and generated demand for HPV vaccine, making it a potential model for HPV immunization in Cameroon and other developing countries.

Assessing the effectiveness of a community-based sensitization strategy in creating awareness about HPV, cervical cancer and HPV vaccine among parents in North West Cameroon.

In 2010, the Cameroon Baptist Convention Health Services (CBCHS) received a donation of HPV vaccine (Gardasil®) to immunize girls of ages 9-13 years in the North West Region of Cameroon. We evaluated the effectiveness of the CBCHS campaign program in sensitizing parents/guardians to encourage HPV vaccine uptake, identified factors that influence parents' decisions to vaccinate girls, and examined the uptake of cervical cancer screening among mothers. We conducted a cross-sectional survey in four healthcare facilities run by CBCHS, churches and other social settings. A total of 350 questionnaires were distributed and 317 were used for the analysis. There were high levels of awareness about cervical cancer, HPV and HPV vaccine. 75.5% understood HPV is sexually transmitted and 90.3% were aware of the use of vaccine as a preventive measure. Effectiveness of the vaccine (31.8%) and side effects/safety (18.4%) were the major barriers for parents to vaccinate their daughters. Bivariate analysis further revealed that the level of education (p = 0.0006), income level (p = 0.0044) and perceived risks (p = 0.0044) are additional factors influencing parents' decisions to vaccinate girls. 35.3% of women had sought a cervical cancer screening, significantly higher than the general estimated rate of screening (<10%) in other parts of Cameroon and sub-Saharan Africa. These results support the viability of a community-tailored sensitization strategy to increase awareness among the targeted audience of parents/guardians, who are critical decision-makers for vaccine delivery to children.

Four Decades of Prophylactic EBV Vaccine Research: A Systematic Review and Historical Perspective.

Background: Epstein-Barr virus (EBV) is the causal agent of infectious mononucleosis and has been associated with various cancers and autoimmune diseases. Despite decades of research efforts to combat this major global health burden, there is no approved prophylactic vaccine against EBV. To facilitate the rational design and assessment of an effective vaccine, we systematically reviewed pre-clinical and clinical prophylactic EBV vaccine studies to determine the antigens, delivery platforms, and animal models used in these studies. Methods: We searched Cochrane Library, ClinicalTrials.gov, Embase, PubMed, Scopus, Web of Science, WHO's Global Index Medicus, and Google Scholar from inception to June 20, 2020, for EBV prophylactic vaccine studies focused on humoral immunity. Results: The search yielded 5,614 unique studies. 36 pre-clinical and 4 clinical studies were included in the analysis after screening against the exclusion criteria. In pre-clinical studies, gp350 was the most commonly used immunogen (33 studies), vaccines were most commonly delivered as monomeric proteins (12 studies), and mice were the most used animal model to test immunogenicity (15 studies). According to an adaptation of the CAMARADES checklist, 4 pre-clinical studies were rated as very high, 5 as high, 13 as moderate quality, 11 as poor, and 3 as very poor. In clinical studies, gp350 was the sole vaccine antigen, delivered in a vaccinia platform (1 study) or as a monomeric protein (3 studies). The present study was registered in PROSPERO (CRD42020198440). Conclusions: Four major obstacles have prevented the development of an effective prophylactic EBV vaccine: undefined correlates of immune protection, lack of knowledge regarding the ideal EBV antigen(s) for vaccination, lack of an appropriate animal model to test vaccine efficacy, and lack of knowledge regarding the ideal vaccine delivery platform. Our analysis supports a multivalent antigenic approach including two or more of the five main glycoproteins involved in viral entry (gp350, gB, gH/gL, gp42) and a multimeric approach to present these antigens. We anticipate that the application of two underused challenge models, rhesus macaques susceptible to rhesus lymphocryptovirus (an EBV homolog) and common marmosets, will permit the establishment of in vivo correlates of immune protection and attainment of more generalizable data. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=198440, identifier PROSPERO I.D. CRD4202019844.

HPV genotyping by L1 amplicon sequencing of archived invasive cervical cancer samples: a pilot study.

BACKGROUND: Human papillomavirus (HPV) is the primary cause of invasive cervical cancer (ICC). The prevalence of various HPV genotypes, ranging from oncogenically low- to high-risk, may be influenced by geographic and demographic factors, which could have critical implications for the screening and prevention of HPV infection and ICC incidence. However, many technical factors may influence the identification of high-risk genotypes associated with ICC in different populations. METHODS: We used high-throughput sequencing of a single amplicon within the HPV L1 gene to assess the influence of patient age, race/ethnicity, histological subtype, sample type, collection date, experimental factors, and computational parameters on the prevalence of HPV genotypes detected in archived DNA (n = 34), frozen tissue (n = 44), and formalin-fixed paraffin-embedded (FFPE) tissue (n = 57) samples collected in the Los Angeles metropolitan area. RESULTS: We found that the percentage of off-target human reads and the concentration of DNA amplified from each sample varied by HPV genotype and by archive type. After accounting for the percentage of human reads and excluding samples with especially low levels of amplified DNA, the HPV prevalence was 95% across all ICC samples: HPV16 was the most common genotype (in 56% of all ICC samples), followed by HPV18 (in 21%). Depending upon the genotyping parameters, the prevalence of HPV58 varied up to twofold in our cohort. In archived DNA and frozen tissue samples, we detected previously established differences in HPV16 and HPV18 frequencies based on histological subtype, but we could not reproduce those findings using our FFPE samples. CONCLUSIONS: In this pilot study, we demonstrate that sample collection, preparation, and analysis methods can influence the detection of certain HPV genotypes and must be carefully considered when drawing any biological conclusions based on HPV genotyping data from ICC samples.

KSHV (HHV8) vaccine: promises and potential pitfalls for a new anti-cancer vaccine.

Seven viruses cause at least 15% of the total cancer burden. Viral cancers have been described as the "low-hanging fruit" that can be potentially prevented or treated by new vaccines that would alter the course of global human cancer. Kaposi sarcoma herpesvirus (KSHV or HHV8) is the sole cause of Kaposi sarcoma, which primarily afflicts resource-poor and socially marginalized populations. This review summarizes a recent NIH-sponsored workshop's findings on the epidemiology and biology of KSHV as an overlooked but potentially vaccine-preventable infection. The unique epidemiology of this virus provides opportunities to prevent its cancers if an effective, inexpensive, and well-tolerated vaccine can be developed and delivered.

Suppression of DC-SIGN and gH Reveals Complex, Subset-Specific Mechanisms for KSHV Entry in Primary B Lymphocytes.

Kaposi sarcoma-associated herpesvirus (KSHV) is the causative agent of multiple cancers in immunocompromised patients including two lymphoproliferative disorders associated with KSHV infection of B lymphocytes. Despite many years of research into the pathogenesis of KSHV associated diseases, basic questions related to KSHV molecular virology remain unresolved. One such unresolved question is the cellular receptors and viral glycoproteins needed for KSHV entry into primary B lymphocytes. In this study, we assess the contributions of KSHV glycoprotein H (gH) and the cellular receptor DC-SIGN to KSHV infection in tonsil-derived B lymphocytes. Our results show that (1) neither KSHV-gH nor DC-SIGN are essential for entry into any B cell subset, (2) DC-SIGN does play a role in KSHV entry into tonsil-derived B cells, but in all B cell subtypes alternative entry mechanisms exist, (3) KSHV-gH can participate in KSHV entry into centrocytes via a DC-SIGN independent entry mechanism, and (4) in the absence of KSHV-gH, DC-SIGN is required for KSHV entry into centrocytes. Our results provide a first glimpse into the complexity of KSHV entry in the lymphocyte compartment and highlight that multiple subset-dependent entry mechanisms are employed by KSHV which depend upon multiple cellular receptors and multiple KSHV glycoproteins.

A Pentavalent Epstein-Barr Virus-Like Particle Vaccine Elicits High Titers of Neutralizing Antibodies against Epstein-Barr Virus Infection in Immunized Rabbits.

Primary infection with Epstein-Barr virus (EBV) is associated with acute infectious mononucleosis, whereas persistent infection is associated with chronic diseases such as autoimmune diseases and various types of cancer. Indeed, approximately 2% of all new cancer cases occurring annually worldwide are EBV-associated. Currently, there is no licensed EBV prophylactic vaccine. Selection of appropriate viral protein subunits is critical for development of an effective vaccine. Although the major EBV surface glycoprotein gp350/220 (gp350) has been proposed as an important prophylactic vaccine target, attempts to develop a potent vaccine based on gp350 alone have shown limited success in the clinic. We provide data showing that five EBV glycoproteins (gp350, gB, gp42, gH, and gL) involved in viral entry and infection can successfully be incorporated on the surface of EBV-like particles (EBV-LPs). These EBV-LPs, when administered together with aluminum hydroxide and monophosphoryl lipid A as adjuvants to New Zealand white rabbits, elicited EBV glycoprotein-specific antibodies capable of neutralizing viral infection in vitro in both B cells and epithelial cells, better than soluble gp350 ectodomain. Our findings suggest that a pentavalent EBV-LP formulation might be an ideal candidate for development as a safe and immunogenic EBV vaccine.

Comparative genomic sequence analysis of novel Helicoverpa armigera nucleopolyhedrovirus (NPV) isolated from Kenya and three other previously sequenced Helicoverpa spp. NPVs.

A newly cloned Helicoverpa armigera nucleopolyhedrovirus (HearNPV) from Kenya, HearNPV-NNg1, has a higher insecticidal activity than HearNPV-G4, which also exhibits lower insecticidal activity than HearNPV-C1. In the search for genes and/or nucleotide sequences that might be involved in the observed virulence differences among Helicoverpa spp. NPVs, the entire genome of NNg1 was sequenced and compared with previously sequenced genomes of G4, C1 and Helicoverpa zea single-nucleocapsid NPV (Hz). The NNg1 genome was 132,425 bp in length, with a total of 143 putative open reading frames (ORFs), and shared high levels of overall amino acid and nucleotide sequence identities with G4, C1 and Hz. Three NNg1 ORFs, ORF5, ORF100 and ORF124, which were shared with C1, were absent in G4 and Hz, while NNg1 and C1 were missing a homologue of G4/Hz ORF5. Another three ORFs, ORF60 (bro-b), ORF119 and ORF120, and one direct repeat sequence (dr) were unique to NNg1. Relative to the overall nucleotide sequence identity, lower sequence identities were observed between NNg1 hrs and the homologous hrs in the other three Helicoverpa spp. NPVs, despite containing the same number of hrs located at essentially the same positions on the genomes. Differences were also observed between NNg1 and each of the other three Helicoverpa spp. NPVs in the diversity of bro genes encoded on the genomes. These results indicate several putative genes and nucleotide sequences that may be responsible for the virulence differences observed among Helicoverpa spp., yet the specific genes and/or nucleotide sequences responsible have not been identified.

Identification of multiple potent neutralizing and non-neutralizing antibodies against Epstein-Barr virus gp350 protein with potential for clinical application and as reagents for mapping immunodominant epitopes.

Prevention of Epstein-Barr virus (EBV) infection has focused on generating neutralizing antibodies (nAbs) targeting the major envelope glycoprotein gp350/220 (gp350). In this study, we generated 23 hybridomas producing gp350-specific antibodies. We compared the candidate gp350-specific antibodies to the well-characterized nAb 72A1 by: (1) testing their ability to detect gp350 using enzyme-linked immunosorbent assay, flow cytometry, and immunoblot; (2) sequencing their heavy and light chain complementarity-determining regions (CDRs); (3) measuring the ability of each monoclonal antibody (mAb) to neutralize EBV infection in vitro; and (4) mapping the gp350 amino acids bound by the mAbs using competitive cell and linear peptide binding assays. We performed sequence analysis to identify 15 mAbs with CDR regions unique from those of murine 72A1 (m72A1). We observed antigen binding competition between biotinylated m72A1, serially diluted unlabeled gp350 nAbs (HB1, HB5, HB11, HB20), and our recently humanized 72A1, but not gp350 non-nAb (HB17) or anti-KSHV gH/gL antibody.

A multivalent Kaposi sarcoma-associated herpesvirus-like particle vaccine capable of eliciting high titers of neutralizing antibodies in immunized rabbits.

Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and the causative agent of multiple cancers in immunocompromised patients. To date, there is no licensed prophylactic KSHV vaccine. In this study, we generated a novel subunit vaccine that incorporates four key KSHV envelope glycoproteins required for viral entry in diverse cell types (gpK8.1, gB, and gH/gL) into a single multivalent KSHV-like particle (KSHV-LP). Purified KSHV-LPs were similar in size, shape, and morphology to KSHV virions. Vaccination of rabbits with adjuvanted KSHV-LPs generated strong glycoprotein-specific antibody responses, and purified immunoglobulins from KSHV-LP-immunized rabbits neutralized KSHV infection in epithelial, endothelial, fibroblast, and B cell lines (60-90% at the highest concentration tested). These findings suggest that KSHV-LPs may be an ideal platform for developing a safe and effective prophylactic KSHV vaccine. We envision performing future studies in animal models that are susceptible to KSHV infection, to determine correlates of immune protection in vivo.