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The duration of cross-neutralising antibody responses (cross-NAb) following HPV immunisation is unknown. We compared cross-NAb responses in cohort of girls who were either unimmunised or had received immunisation with one, two or three doses of 4vHPV (Gardasil&reg, Merck Inc.) six years earlier, before and one month after a booster dose of 2vHPV (Cervarix&reg, GSK). NAb to potentially cross-reactive HPV genotypes 31, 33, 45, 52 and 58 were measured using a HPV pseudovirion-based neutralisation assay. Girls who had previously received at least one dose of 4vHPV had significantly higher NAb titres for HPV31 when compared with unimmunised girls, whereas no difference in NAb titre was observed for four other genotypes (33, 45, 52 and 58). Following a single further immunisation with 2vHPV, NAb titres to each of the five tested HPV genotypes were comparable for girls who previously received one, two or three doses of 4vHPV, and were significantly higher than for previously unimmunised girls. Immunisation with one, two or three doses of 4vHPV induced NAb to HPV31 that persisted for six years, but there was no persistence of NAb to HPV33, 45, 52 or 58. Our results suggest that one or two doses of 4vHPV may provide long-term protection against HPV31.

The use of plants for the production of virus-like nanoparticles (VNPs) dates back to separating natural empty capsids of plant viruses from whole virions nearly 70 years ago, through to the present use of transgenic plants or recombinant Agrobacterium tumefaciens and/or plant virus-derived vectors for the transient expression of engineered viral or other structural proteins in plants-a production system also known as molecular farming. Plant production of heterologous proteins has major advantages in terms of convenience-whole plants are generally used, and processes do not need to be sterile-and cost, as bulk biomass production is significantly cheaper than by any other method. Plant-made VNPs in current use for nanotechnology include whole virions and naturally occurring empty capsids of plant viruses, and particles made by reassembly of coat protein (CP) purified from virions or by recombinant expression. Engineered VNP-forming animal or human virus CPs expressed in plants include L1 protein from human papillomaviruses, human norovirus CP, hepatitis B surface and core antigens, influenza virus HA protein and HIV Gag polyprotein forming large enveloped particles by budding, orbi- and rotavirus particles that require assembly of four co-expressed proteins, and polio- and foot and mouth disease viruses which require proteolytic processing of a polyprotein precursor to form 4-component VNPs. Both plant and animal virus-derived plant-made VNPs can be used for surface and internal display of heterologous peptides or even whole proteins. A significant recent development has been the production of pseudovirions in plants, comprising plant or animal virus CPs and RNA or DNA pseudogenomes that can be used to deliver nucleic acid payloads into cultured cells or specific tissues or tumors in whole animals. This article is characterized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Therapeutic Approaches and Drug Discovery > Emerging Technologies Diagnostic Tools > in vivo Nanodiagnostics and Imaging.

Cervical cancer caused by infection with human papillomaviruses (HPVs) is the fourth most common cancer in women globally, with the burden mainly in developing countries due to limited healthcare resources. Current vaccines based on virus-like particles (VLPs) assembled from recombinant expression of the immunodominant L1 protein are highly effective in the prevention of cervical infection; however, these vaccines are expensive and type-specific. Therefore, there is a need for more broadly protective and affordable vaccines. The HPV-16 L2 peptide sequences 108-120, 65-81, 56-81, and 17-36 are highly conserved across several HPV types and have been shown to elicit cross-neutralizing antibodies. To increase L2 immunogenicity, L1:L2 chimeric VLPs (cVLP) vaccine candidates were developed. The four L2 peptides mentioned above were substituted into the DE loop of HPV-16 L1 at position 131 (SAC) or in the C-terminal region at position 431 (SAE) to generate HPV-16-derived L1:L2 chimeras. All eight chimeras were transiently expressed in Nicotiana benthamiana via Agrobacterium tumefaciens-mediated DNA transfer. SAC chimeras predominantly assembled into higher order structures (T = 1 and T = 7 VLPs), whereas SAE chimeras assembled into capsomeres or formed aggregates. Four SAC and one SAE chimeras were used in vaccination studies in mice, and their ability to generate cross-neutralizing antibodies was analyzed in HPV pseudovirion-based neutralization assays. Of the seven heterologous HPVs tested, cross-neutralization with antisera specific to chimeras was observed for HPV-11 (SAE 65-18), HPV-18 (SAC 108-120, SAC 65-81, SAC 56-81, SAE 65-81), and HPV-58 (SAC 108-120). Interestingly, only anti-SAE 65-81 antiserum showed neutralization of homologous HPV-16, suggesting that the position of the L2 epitope display is critical for maintaining L1-specific neutralizing epitopes.

Human Immunodeficiency virus (HIV) virus-like Particles (VLPs) composed of HIV(IIIB) Gag and HIV(BaL) gp120/gp41 envelope are a pseudovirus vaccine capable of presenting antigens in their native conformations. To enhance the immunogenicity of the HIV Env antigen, VLPs were coupled to VesiVax Conjugatable Adjuvant Lipid Vesicles (CALV) containing one of four toll-like-receptor (TLR) ligands, each activating a receptor with distinct cellular localization and downstream pathways. C57Bl/6 mice were vaccinated by intranasal prime followed by two sub-cheek boosts and their sera immunoglobulin and neutralizing potency were measured over a duration of 3 months after vaccination. PBS control, VLPs alone, CALV + VLPs, and VLPs complexed with CALV and ligands for TLR2 (PAM3CAG), TLR3 (dsRNA), TLR4 (MPLA), and TLR7/8 (resiquimod) were evaluated based on antibody titer, IgG1 and IgG2c class switching, germinal center formation, T follicular cells and potency of neutralizing antibodies. Consistently, the TLR3 ligand dsRNA complexed to CALV and in combination with VLPs (CALV(dsRNA)+VLPs) induced the strongest response. CALV(dsRNA)+VLPs induced the highest titers against the recombinant vaccine antigens clade B Bal gp120 and pr55 Gag. Additionally, CALV(dsRNA)+VLPs induced cross-clade antibodies, represented by high titers of antibody to clade c 96ZM651 gp120. CALV(dsRNA)+VLPs induced predominantly IgG2c over IgG1, a response associated with T helper type 1 (Th1)-like cytokines. In turn, CALV(dsRNA)+VLP immunized mice generated the most potent neutralizing antibodies against HIV strain MN.3. Finally, at time of sacrifice, a significant increase in germinal center B cells and T follicular cells was detected in mice which received CALV(dsRNA)+VLPs compared to PBS. Our results indicate that CALV(dsRNA) is a superior adjuvant for HIV VLPs in generating a Th1-like immunoglobulin profile, while prolonging lymph node germinal centers, T follicular cells and generating neutralizing antibodies to a highly sensitive tier 1A variant of HIV.

Human papillomavirus (HPV) 6 is a human pathogen which causes genital warts. Recombinant virus-like particle (VLP) based antigens are the active components in prophylactic vaccines to elicit functional antibodies. The binding and functional characteristics of a panel of 15 murine monoclonal antibodies (mAbs) against HPV6 was quantitatively assessed. Elite conformational indicators, recognizing the conformational epitopes, are also elite viral neutralizers as demonstrated with their viral neutralization efficiency (5 mAbs with neutralization titer below 4ng/mL) in a pseudovirion (PsV)-based system. The functionality of a given mAb is closely related to the nature of the corresponding epitope, rather than the apparent binding affinity to antigen. The epitope-specific antigenicity assays can be used to assess the binding activity of PsV or VLP preparations to neutralizing mAbs. These mAb-based assays can be used for process monitoring and for product release and characterization to confirm the existence of functional epitopes in purified antigen preparations. Due to the particulate nature of the alum adjuvants, the vaccine antigen adsorbed on adjuvants was considered largely as "a black box" due to the difficulty in analysis and visualization. Here, a novel method with fluorescence-based high content imaging for visualization and quantitating the immunoreactivity of adjuvant-adsorbed VLPs with neutralizing mAbs was developed, in which antigen desorption was not needed. The facile and quantitative in situ antigenicity analysis was amendable for automation. The integrity of a given epitope or two non-overlapping epitopes on the recombinant VLPs in their adjuvanted form can be assessed in a quantitative manner for cross-lot or cross-product comparative analysis with minimal manipulation of samples.

Background Most cervical cancers are caused by vaccine-preventable infections with human papillomaviruses (HPV). The HPV prophylactic vaccines Gardasil and Cervarix have been widely used for >10 years and are reported to induce high antibody levels. A head-to-head comparison of the antibody responses induced by the 2 vaccines has been performed only up to 5 years. Methods Among 3300 Finnish females aged 16-17 years who got 1 of the 2 HPV vaccines in phase 3 licensure trials, virtually all consented to registry-based long-term follow-up. Linkage with the Finnish Maternity Cohort found that they donated >2500 serum samples up to 12 years later. Sera of 337 (38.6%) Gardasil and 730 (30.3%) Cervarix vaccine recipients were retrieved from the Finnish Maternity Cohort biobank and type-specific anti-HPV antibody levels were determined using in-house multiplexed heparin-HPV pseudovirion Luminex assay. Results Anti-HPV-16 and anti-HPV-18 antibody levels remained stable and above natural infection-related antibody levels for up to 12 years for most vaccine recipients. The median antibody levels were higher among Cervarix recipients 7-12 years post vaccination (P < .0001). Conclusions The stability of vaccine-induced antibody levels is in accordance with the high long-term protection reported previously. The differences in antibody levels induced by the 2 vaccines imply that continued follow-up to identify possible breakthrough cases and estimation of the minimal protective levels of serum antibodies is a research priority.

Inimese papilloomiviiruste kvaasi-ja pseudovirionide konstrueerimine ning nakatamisvõime analüüsimine U2OS ja 293FT rakuliinides. Papilloomiviirused on väikesed DNA viirused, mis nakatavad epiteelkoe keratinotsüüte ja nende elutsükkel on tihedalt seotud epiteelkoe rakkude jagunemisega. Üks võimalus papilloomiviiruste uurimiseks ning nakatamisvõimeliste virionide saamiseks laboritingimustes on valmistada sünteetilisi viiruspartikleid. Käesolev bakalaureusetöö keskendub HPV11 genoomi ja HPV18 varajasi avatud lugemisraame sisaldavate kvaasivirionide ning GFP-d ekspresseerivate pseudovirionide valmistamisele ning nende nakatamisvõime hindamisele U2OS ja FT293 rakkudes.

Persistent high-risk human papillomavirus (HPV) infection is linked to cervical cancer. Two prophylactic virus-like particle (VLP)-based vaccines have been marketed globally for nearly a decade. Here, we review the HPV pseudovirion (PsV)-based assays for the functional assessment of the HPV neutralizing antibodies and the structural basis for these clinically relevant epitopes. The PsV-based neutralization assay was developed to evaluate the efficacy of neutralization antibodies in sera elicited by vaccination or natural infection or to assess the functional characteristics of monoclonal antibodies. Different antibody binding modes were observed when an antibody was complexed with virions, PsVs or VLPs. The neutralizing epitopes are localized on surface loops of the L1 capsid protein, at various locations on the capsomere. Different neutralization antibodies exert their neutralizing function via different mechanisms. Some antibodies neutralize the virions by inducing conformational changes in the viral capsid, which can result in concealing the binding site for a cellular receptor like 1A1D-2 against dengue virus, or inducing premature genome release like E18 against enterovirus 71. Higher-resolution details on the epitope composition of HPV neutralizing antibodies would shed light on the structural basis of the highly efficacious vaccines and aid the design of next generation vaccines. In-depth understanding of epitope composition would ensure the development of function-indicating assays for the comparability exercise to support process improvement or process scale up. Elucidation of the structural elements of the type-specific epitopes would enable rational design of cross-type neutralization via epitope re-engineering or epitope grafting in hybrid VLPs.

Introduction: Cervical cancer is the second most common cancer in women worldwide and the role of human papillomavirus (HPV) has been proved in its etiology. The currentley available L1-capsid-protein-based vaccine is highly immunogenic and very high titers of serum antibodies can be obtained by its injection, but unfortunately it is restricted to only a few HPV genotypes and is relatively expensive. Therefore, development of the second-generation HPV vaccines has become the focus of the research and L2 capsid protein that is capable of producing broad spectrum antibodies has become one of the main candidates. Evaluation of the vaccine immunogenicity however, requires develoment of HPV pseudovirions (HPV PvSs) comprised of L1 and L2 virus protein and the present study was an attempt to produce PvSs of HPV18 genotype by an in-house method. Methods: The HPV18 L1/L2 coding plasmid and the reporter plasmid of pEGFP-N1 were amplified in E. coli DH5α and purified using silica oxide method. The plasmids were co-transfected into HEK 293FT cell line and the preliminary analysis of expression was performed using fluorescence microscopy. The PvSs were partially purified using gel filtration chromatography and were used to transduce the 293FT cells to evaluate the infectivity rate of the PvSs. The results were analyzed by fluorescent microscopy, flow cytometry and atomic force microscopy. Results: The results showed that the HPV18 L1/L2 coding plasmid and the pEGFP-N1 reporter plasmid have been successfully co-transfected into HEK 293FT cells and the PvSs were constructed. The 293FT cells were successfully transduced by PvSs that had packaged reporter plasmid. These findings were confirmed by fluorescence microscopy and flow cytometry as well as AFM imaging. Conclusion: In this study, the cotransfection of HPV18 L1/L2 coding plasmid as well as pEGP-N1 reporter plasmid into the HEK 293FT cell led to the assembly of the pseudovirion harboring the reporter gene. The protocols used in this study were easy to perform and relatively inexpensive and did not rely on the commercial kits.

Ebolavirus can cause hemorrhagic fever in humans with a mortality rate of 50%–90%. Currently, no approved vaccines and antiviral therapies are available. Human TIM1 is considered as an attachment factor for EBOV, enhancing viral infection through interaction with PS located on the viral envelope. However, reasons underlying the preferable usage of hTIM-1, but not other PS binding receptors by filovirus, remain unknown. We firstly demonstrated a direct interaction between hTIM-1 and EBOV GP in vitro and determined the crystal structures of the Ig V domains of hTIM-1 and hTIM-4. The binding region in hTIM-1 to EBOV GP was mapped by chimeras and mutation assays, which were designed based on structural analysis. Pseudovirion infection assays performed using hTIM-1 and its homologs as well as point mutants verified the location of the GP binding site and the importance of EBOV GP-hTIM-1 interaction in EBOV cellular entry. Electronic supplementary material The online version of this article (doi:10.1007/s13238-015-0220-y) contains supplementary material, which is available to authorized users.

The infectious process of human papillomaviruses (HPVs) has been studied considerably, and many cellular components required for viral entry and trafficking continue to be revealed. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during HPV16 pseudovirion infection of human keratinocytes. We found that Pyk2 is necessary for infection and appears to be involved in the intracellular trafficking of the virus. Small interfering RNA-mediated reduction of Pyk2 resulted in a significant decrease in infection but did not prevent viral entry at the plasma membrane. Pyk2 depletion resulted in altered endolysosomal trafficking of HPV16 and accelerated unfolding of the viral capsid. Furthermore, we observed retention of the HPV16 pseudogenome in the trans -Golgi network (TGN) in Pyk2-depleted cells, suggesting that the kinase could be required for the viral DNA to exit the TGN. While Pyk2 has previously been shown to function during the entry of enveloped viruses at the plasma membrane, the kinase has not yet been implicated in the intracellular trafficking of a nonenveloped virus such as HPV. Additionally, these data enrich the current literature on Pyk2's function in human keratinocytes. IMPORTANCE In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during human papillomavirus (HPV) infection of human skin cells. Infections with high-risk types of HPV such as HPV16 are the leading cause of cervical cancer and a major cause of genital and oropharyngeal cancer. As a nonenveloped virus, HPV enters cells by interacting with cellular receptors and established cellular trafficking routes to ensure that the viral DNA reaches the nucleus for productive infection. This study identified Pyk2 as a cellular component required for the intracellular trafficking of HPV16 during infection. Understanding the infectious pathways of HPVs is critical for developing additional preventive therapies. Furthermore, this study advances our knowledge of intracellular trafficking processes in keratinocytes.

Previous studies have indicated that human papillomavirus (HPV) infectious entry is slow, requiring many hours after initial infection for the virus to gain entry into the nucleus. However, intracellular transport pathways typically are very rapid, and in the context of a natural HPV infection in a wounded epithelium, such slow intracellular transport would seem to be at odds with a normal viral infection. Using synchronized cell populations, we show that HPV trafficking can be a rapid process. In cells that are infected in the late S-early G 2 /M phase of the cell cycle, HPV16 pseudovirion (PsV) reporter DNA gene expression is detectable by 8 h postinfection. Likewise, reporter DNA can be visualized within the nucleus in conjunction with PML nuclear bodies 1 h to 2 h postinfection in cells that are infected with PsVs just prior to mitotic entry. This demonstrates that endosomal trafficking of HPV is rapid, with mitosis being the main restriction on nuclear entry. IMPORTANCE HPV infectious entry appears to be slow and requires mitosis to occur before the incoming viral DNA can access the nucleus. In this study, we show that HPV trafficking in the cell actually is very rapid. This demonstrates that in the context of a normal virus infection, the cell cycle state will have a major influence on the time it takes for an incoming virus to enter the nucleus and initiate viral gene expression.

To understand and compare the mechanisms of murine and human PV infection, we examined pseudovirion binding and infection of the newly described MusPV1 using the murine cervicovaginal challenge model. These analyses revealed primary tissue interactions distinct from those previously described for HPV16. Unlike HPV16, MusPV1 bound basement membrane (BM) in an HSPG-independent manner. Nevertheless, subsequent HSPG interactions were critical. L2 antibodies or low doses of VLP antibodies, sufficient to prevent infection, did not lead to disassociation of the MusPV1 pseudovirions from the BM, in contrast to previous findings with HPV16. Similarly, furin inhibition did not lead to loss of MusPV1 from the BM. Therefore, phylogenetically distant PV types differ in their initial interactions with host attachment factors, but initiate their lifecycle on the acellular BM. Despite these differences, these distantly related PV types displayed similar intracellular trafficking patterns and susceptibilities to biochemical inhibition of infection.

To assess immunogenicity and development of antibodies in the context of vaccination, it is critical to quantify titers of neutralizing antibodies. We have been employing the 293TT cell-based neutralization assay system to quantify anti-HPV neutralizing antibodies. In this system, human papillomavirus (HPV) pseudovirion (PsV) particles encapsidating secreted alkaline phosphatase (SEAP) gene are used to measure infection of 293TT cells in 72-hr cell-culture supernatants. SEAP has traditionally been measured by Great EscAPe™ SEAP Chemiluminescence Kit 2.0 (GE). To reduce the cost, and to potentially increase efficiency, we sought a cheaper kit with better detection capability. Performance characteristics of the newer chemiluminescence kit, ZiVa® Ultra SEAP Plus Assay (Ziva) and GE were compared using the 293TT system. Dose titration of HPV PsV 16 or 18 showed that signal-to-noise ratios at 48 and 72 hr post-infection were higher for ZiVa at nearly all doses. ZiVa was superior to GE as it was able to detect SEAP at 48 hr, as well as when lower numbers of 293TT cells were used. The ability of ZiVa to quantitate HPV-16 and -18 neutralizing antibody titers was tested using sera from Cervarix® immunized individuals. Spearman rank correlational analyses showed excellent correlations between the titers obtained with ZiVa and GE for anti-HPV16 (r = 0.9822, p < 0.0001) and anti-HPV18 (r = 0.9832, p < 0.0001) antibodies. We concluded that ZiVa is superior to GE in detecting SEAP, and the antibody titers in sera of vaccinated individuals were similar to those obtained with GE. Thus, Ziva is a suitable alternative to GE.

Members of the family Filoviridae are filamentous, enveloped, and nonsegmented negative-stranded RNA viruses that can cause severe hemorrhagic disease in humans and nonhuman primates with high mortality rates. Current efforts to analyze the structure and biology of these viruses as well as the development of antivirals have been hindered by the necessity of biosafety level 4 containment (BSL4). Here, we outline how to produce and work with Ebola virus glycoprotein bearing vesicular stomatitis virus (VSV) pseudovirions. These pseudovirions can be safely used to evaluate early steps of the filovirus life cycle without need for BSL4 containment. Virus gene expression in the transduced cells is easy to assess since the pseudovirions encode a reporter gene in place of the VSV G glycoprotein gene. Adoption of VSV for use as a pseudovirion system for filovirus GP has significantly expanded access for researchers to study specific aspects of the viral life cycle outside of BSL4 containment and has allowed substantial growth of filovirus research.

The recent Ebola virus (EBOV) epidemic in West Africa demonstrates the potential for a significant public health burden caused by filoviral infections. No vaccine or antiviral is currently FDA approved. To expand the vaccine options potentially available, we assessed protection conferred by an EBOV vaccine composed of vesicular stomatitis virus pseudovirions that lack native G glycoprotein (VSVΔG) and bear EBOV glycoprotein (GP). These pseudovirions mediate a single round of infection. Both single-dose and prime/boost vaccination regimens protected mice against lethal challenge with mouse-adapted Ebola virus (ma-EBOV) in a dose-dependent manner. The prime/boost regimen provided significantly better protection than a single dose. As N-linked glycans are thought to shield conserved regions of the EBOV GP receptor-binding domain (RBD), thereby blocking epitopes within the RBD, we also tested whether VSVΔG bearing EBOV GPs that lack GP1 N-linked glycans provided effective immunity against challenge with ma-EBOV or a more distantly related virus, Sudan virus. Using a prime/boost strategy, high doses of GP/VSVΔG partially or fully denuded of N-linked glycans on GP1 protected mice against ma-EBOV challenge, but these mutants were no more effective than wild-type (WT) GP/VSVΔG and did not provide cross protection against Sudan virus. As reported for other EBOV vaccine platforms, the protection conferred correlated with the quantity of EBOV GP-specific Ig produced but not with the production of neutralizing antibodies. Our results show that EBOV GP/VSVΔG pseudovirions serve as a successful vaccination platform in a rodent model of Ebola virus disease and that GP1 N-glycan loss does not influence immunogenicity or vaccination success. IMPORTANCE The West African Ebola virus epidemic was the largest to date, with more than 28,000 people infected. No FDA-approved vaccines are yet available, but in a trial vaccination strategy in West Africa, recombinant, infectious VSV encoding the Ebola virus glycoprotein effectively prevented virus-associated disease. VSVΔG pseudovirion vaccines may prove as efficacious and have better safety, but they have not been tested to date. Thus, we tested the efficacy of VSVΔG pseudovirions bearing Ebola virus glycoprotein as a vaccine platform. We found that wild-type Ebola virus glycoprotein, in the context of this platform, provides robust protection of EBOV-challenged mice. Further, we found that removal of the heavy glycan shield surrounding conserved regions of the glycoprotein does not enhance vaccine efficacy.

Nat. Med.; 10.1038/nm.4201; corrected online 15 November 2016 In the version of this article initially published online, the authors omitted acknowledging a funding agency and researchers for providing the pseudovirion neutralization assay data. The acknowledgments section has been revised to state,“We thank the US Army Medical Research Institute of Infectious Diseases team, including S.

Common cutaneous human papillomavirus (HPV) types induce skin warts, whereas species beta HPV are implicated, together with UV-radiation, in the development of non-melanoma skin cancer (NMSC) in immunosuppressed patients. Licensed HPV vaccines contain virus-like particles (VLP) self-assembled from L1 major capsid proteins that provide type-restricted protection against mucosal HPV infections causing cervical and other ano-genital and oro-pharyngeal carcinomas and warts (condylomas), but do not target heterologous HPV. Experimental papillomavirus vaccines have been designed based on L2 minor capsid proteins that contain type-common neutralization epitopes, to broaden protection to heterologous mucosal and cutaneous HPV types. Repetitive display of the HPV16 L2 cross-neutralization epitope RG1 (amino acids (aa) 17-36) on the surface of HPV16 L1 VLP has greatly enhanced immunogenicity of the L2 peptide. To more directly target cutaneous HPV, L1 fusion proteins were designed that incorporate the RG1 homolog of beta HPV17, the beta HPV5 L2 peptide aa53-72, or the common cutaneous HPV4 RG1 homolog, inserted into DE surface loops of HPV1, 5, 16 or 18 L1 VLP scaffolds. Baculovirus expressed chimeric proteins self-assembled into VLP and VLP-raised NZW rabbit immune sera were evaluated by ELISA and L1- and L2-based pseudovirion (PsV) neutralizing assays, including 12 novel beta PsV types. Chimeric VLP displaying the HPV17 RG1 epitope, but not the HPV5L2 aa53-72 epitope, induced cross-neutralizing humoral immune responses to beta HPV. In vivo cross-protection was evaluated by passive serum transfer in a murine PsV challenge model. Immune sera to HPV16L1-17RG1 VLP (cross-) protected against beta HPV5/20/24/38/96/16 (but not type 76), while antisera to HPV5L1-17RG1 VLP cross-protected against HPV20/24/96 only, and sera to HPV1L1-4RG1 VLP cross-protected against HPV4 challenge. In conclusion, RG1-based VLP are promising next generation vaccine candidates to target cutaneous HPV infections.

HPV-023 (NCT00518336; ClinicalTrial.gov) is a long-term follow-up of an initial double-blind, randomized (1:1), placebo-controlled study (HPV-001, NCT00689741) evaluating the efficacy against human papillomavirus (HPV)-16/18 infection and associated cyto-histopathological abnormalities, persistence of immunogenicity, and safety of the HPV-16/18 AS04-adjuvanted vaccine. Among the women, aged 15–25 years, enrolled in HPV-001 and who participated in the follow-up study HPV-007 (NCT00120848), a subset of 437 women from five Brazilian centers participated in this 36-month long-term follow-up (HPV-023) for a total of 113 months (9.4 years). During HPV-023, anti-HPV-16/18 antibodies were measured annually by enzyme-linked immunosorbent assay (ELISA) and pseudovirion-based neutralisation assay (PBNA). Cervical samples were tested for HPV DNA every 6 months, and cyto-pathological examinations were performed annually. During HPV-023, no new HPV-16/18-associated infections and cyto-histopathological abnormalities occurred in the vaccine group. Vaccine efficacy (VE) against HPV-16/18 incident infection was 100% (95%CI: 66.1, 100). Over the 113 months (9.4 years), VE was 95.6% (86.2, 99.1; 3/50 cases in vaccine and placebo groups, respectively) against incident infection, 100% (84·1, 100; 0/21) against 6-month persistent infection (PI); 100% (61·4, 100; 0/10) against 12-month PI; 97·1% (82.5, 99.9; 1/30) against ≥ ASC-US; 95·0% (68.0, 99.9; 1/18) against ≥ LSIL; 100% (45.2, 100; 0/8) against CIN1+; and 100% (–128.1, 100; 0/3) against CIN2+ associated with HPV-16/18. All vaccinees remained seropositive to HPV-16/18, with antibody titers remaining several folds above natural infection levels, as measured by ELISA and PBNA. There were no safety concerns. To date, these data represent the longest follow-up reported for a licensed HPV vaccine.

Retroviral and lentiviral vector pseudotypes (based on human immunodeficiency virus type 1, HIV-1) have been used for stable and safe gene transfer because of their broad host ranges and high mechanical strength. In the present study, a recombinant avian leukosis virus subgroup J (ALV-J) polypeptide pseudotyped with lentivirus membrane glycoproteins gp85 and gp37, HIV/env-ALV, was generated, characterized in vitro and evaluated for its ability to infect natural host cells. We optimized the newly developed micro-neutralization (MN) assay using recombinant pseudovirion HIV/env-ALV expressing enhanced green fluorescent protein and well-characterized sera from chickens with confirmed ALV-J disease or virus-free controls. HIV/env-ALV could infect CEF and DF-1 but not pk15, 293FT, MDCK or VERO E6 cells, therefore demonstrating a cellular tropism similar to the wild-type ALV-J. The MN assay indicated that the IC50 values of positive sera offered a considerable advantage in both speed and accuracy. These results suggest that this pseudotyped lentivirus is a good model for studying the functions of ALV-J env and that the MN assay is a reliable serological method for assessing antibody levels in investigating the actual status of the current ALV-J epidemic. These recombinant pseudovirions may prove to be useful for studying ALV-J biology in lower biosafety level laboratory environments, and also for the detection and quantification of neutralizing antibodies to ALV-J in a manner akin to ELISA assays, but that would also be applicable to other viruses.

Human papillomavirus (HPV) is the well-known second most cause of cervical cancer in women worldwide. According to the WHO survey, 70% of the total cervical cancers are associated with types HPV 16 and 18. Presently used prophylactic vaccine for HPV contains mainly capsid protein of L1 virus like particles (VLPs). Correct folding of VLPs and display of neutralizing epitopes are the major constraint for VLP-based vaccines. Further, monoclonal antibodies (mAbs) play a vital role in developing therapeutics and diagnostics. mAbs are also useful for the demonstration of VLP conformation, virus typing and product process assessment as well. In the present study, we have explored the usefulness of mAbs generated against sf-9 expressed HPV 16 VLPs demonstrated as type-specific and conformational dependent against HPV 16 VLPs by ELISA. High affinity and high pseudovirion neutralization titer of mAbs indicated their potential for the development of prophylactic vaccines for HPV. Also, the type-specific and conformational reactivity of the mAbs to HPV 16 VLPs in sf-9 cells by immunofluorescence assay proved their diagnostic potential.

Mouse polyomavirus (MPyV) is considered a potential tool for the application of gene therapy; however, the current knowledge of the encapsulation of DNA into virions is vague. We used a series of assays based on the encapsidation of a reporter vector into MPyV pseudovirions to identify putative cis-acting elements that are involved in DNA encapsidation. None of the sequences that were derived from MPyV have been shown to solely enhance the encapsidation of a reporter vector in the assay. The frequency of encapsidation strongly correlated with the total intracellular amount of the vector after transfection. The encapsidation of target DNA into the pseudovirions was shown to be non-specific, and the packaging of non-replicated DNA was observed. We propose that the actual concentration of target DNA at the sites of virion formation is the primary factor that determines its selection for encapsidation.

A pseudovirion-based neutralisation assay (PBNA) has been considered the gold standard for measuring specific antibody responses against human papillomavirus (HPV). However, this assay is labor intensive and therefore very difficult to implement in large-scale studies. Previous studies have evaluated the agreement between virus-like particle (VLP)-based ELISA and PBNA for measuring HPV vaccine-induced antibodies. However, the concordance of these assays to detect antibodies induced by natural infection has not yet been fully elucidated. In this study, the results of an Escherichia coli (E. coli)-expressed VLP-based ELISA were found to be highly concordant with those of a baculovirus-expressed VLP-based ELISA (r = 0.96 and 0.97 for HPV-16 and HPV-18) when detecing HPV vaccine induced antibodies and the concordance was medium (r = 0.68 and 0.68 for HPV-16 and HPV-18) when assessing natural infection induced antibodies. The results of the E. coli expressed VLP-based ELISA correlated well with those of the PBNA when testing 1020 post-vaccination human sera collected at one month after vaccination with the E. coli expressed VLP-based bivalent HPV vaccine (r = 0.83 and 0.81 for HPV-16 and HPV-18). The agreement and correlation were moderate (kappa < 0.3 for both HPV types 16 and 18, r = 0.59 and 0.68 for HPV-16 and HPV-18, respectively) when assessing 1600 serum samples from unvaccinated women of age 18–25 years. In conclusion, the VLP-based ELISA is an acceptable surrogate for the neutralizing antibody assay in measuring vaccine responses. However, the use of the VLP-based ELISA in epidemiological studies should be carefully considered.

Virus-like particles (VLPs), aggregates of capsid proteins devoid of viral genetic material, show great promise in the fields of vaccine development and gene therapy. These particles spontaneously self-assemble after heterologous expression of viral structural proteins. This review will focus on the use of virus-like particles derived from polyomavirus capsid proteins. Since their first recombinant production 27 years ago these particles have been investigated for a myriad of biomedical applications. These virus-like particles are safe, easy to produce, can be loaded with a broad range of diverse cargoes and can be tailored for specific delivery or epitope presentation. We will highlight the structural characteristics of polyomavirus-derived VLPs and give an overview of their applications in diagnostics, vaccine development and gene delivery.

The cell surface receptor T cell immunoglobulin mucin domain 1 (TIM-1) dramatically enhances filovirus infection of epithelial cells. Here, we showed that key phosphatidylserine (PtdSer) binding residues of the TIM-1 IgV domain are critical for Ebola virus (EBOV) entry through direct interaction with PtdSer on the viral envelope. PtdSer liposomes but not phosphatidylcholine liposomes competed with TIM-1 for EBOV pseudovirion binding and transduction. Further, annexin V (AnxV) substituted for the TIM-1 IgV domain, supporting a PtdSer-dependent mechanism. Our findings suggest that TIM-1-dependent uptake of EBOV occurs by apoptotic mimicry. Additionally, TIM-1 enhanced infection of a wide range of enveloped viruses, including alphaviruses and a baculovirus. As further evidence of the critical role of enveloped-virion-associated PtdSer in TIM-1-mediated uptake, TIM-1 enhanced internalization of pseudovirions and virus-like proteins (VLPs) lacking a glycoprotein, providing evidence that TIM-1 and PtdSer-binding receptors can mediate virus uptake independent of a glycoprotein. These results provide evidence for a broad role of TIM-1 as a PtdSer-binding receptor that mediates enveloped-virus uptake. Utilization of PtdSer-binding receptors may explain the wide tropism of many of these viruses and provide new avenues for controlling their virulence.

Prophylactic vaccines against human papillomavirus (HPV) based on virus-like particles (VLP) induce type-specific neutralizing antibodies against a small number of hypervariable residues positioned in surface-exposed loops of the major capsid protein L1. To investigate the importance of these residues for neutralization, cross-neutralization, L2 incorporation and genome encapsidation, ten surface-exposed amino acid residues in four hypervariable loops of L1 were mutated. VLPs containing mutated or WT L1, with or without WT L2, were produced in 293TT cells using pseudovirion expression vectors. The mutations reduced the ability to induce neutralizing antibodies and to incorporate the L2 protein in the capsid. Ability to induce cross-neutralizing antibodies and to encapsidate pseudogenomes were completely abrogated. In summary, the surface-exposed L1 loops are important for the function of the HPV particle.

Licensed human papillomavirus (HPV) vaccines, based on virus-like particles (VLPs) self-assembled from major capsid protein L1, afford type-restricted protection against HPV types 16/18/6/11 (or 16/18 for the bivalent vaccine), which cause 70% of cervical cancers (CxCas) and 90% of genital warts. However, they do not protect against less prevalent high-risk (HR) types causing 30% of CxCa, or cutaneous HPV. In contrast, vaccination with the minor capsid protein L2 induces low-level immunity to type-common epitopes. Chimeric RG1-VLP presenting HPV16 L2 amino acids 17–36 (RG1 epitope) within the DE-surface loop of HPV16 L1 induced cross-neutralizing antisera. We hypothesized that RG1-VLP vaccination protects against a large spectrum of mucosal and cutaneous HPV infections in vivo. Immunization with RG1-VLP adjuvanted with human-applicable alum-MPL (aluminum hydroxide plus 3-O-desacyl-4′-monophosphoryl lipid A) induced robust L2 antibodies (ELISA titers 2,500–12,500), which (cross-)neutralized mucosal HR HPV16/18/45/37/33/52/58/35/39/51/59/68/73/26/69/34/70, low-risk HPV6/11/32/40, and cutaneous HPV2/27/3/76 (titers 25–1,000) using native virion- or pseudovirion (PsV)-based assays, and a vigorous cytotoxic T lymphocyte response by enzyme-linked immunospot. In vivo, mice were efficiently protected against experimental vaginal challenge with mucosal HR PsV types HPV16/18/45/31/33/52/58/35/39/51/59/68/56/73/26/53/66/34 and low-risk HPV6/43/44. Enduring protection was demonstrated 1 year after vaccination. RG1-VLP is a promising next-generation vaccine with broad efficacy against all relevant mucosal and also cutaneous HPV types.

Purpose: Cancer immunotherapy has encountered many difficulties in the face of the expectation to eradicate cancer, and new breakthroughs are required. We have previously shown that UV-inactivated Sendai virus particles (hemagglutinating virus of Japan envelope; HVJ-E) induce immunity against multiple tumor types. In this study, a novel pseudovirion that stimulates more robust antitumor immunity was designed for cancer treatment. Experimental Design: First, we found that culturing murine splenocytes with HVJ-E in combination with interleukin (IL)-12 resulted in a remarkable increase in IFN-γ production compared with that observed in splenocytes cultured with IL-12 alone. The synergistic effects of HVJ-E and IL-12 on IFN-γ production were caused by viral F proteins independently of HVJ-E fusion activity and not by hemagglutination from hemagglutinin-neuraminidase (HN) proteins. We next constructed HN-depleted HVJ-E expressing the Fc region of immunoglobulin G (IgG) on the envelope and single-chain IL-12 containing the ZZ domain of protein A to produce an IL-12–conjugated HVJ-E particle without hemagglutinating activity. Results: IL-12–conjugated HVJ-E dramatically enhanced the amount of IFN-γ produced by immune cells. Intratumoral injection of IL-12–conjugated HVJ-E eradicated murine melanomas more effectively than injection of wild-type HVJ-E through increased production of melanoma-specific CTLs. IL-12–conjugated HVJ-E preferentially accumulated in the lungs after systemic administration. When small metastatic melanoma foci were formed in the lungs, systemic administration of IL-12–conjugated HVJ-E significantly reduced the number of metastatic foci by inducing local production of IFN-γ in the lungs and generating large numbers of melanoma-specific CTLs. Conclusion: IL-12–conjugated HVJ-E is a promising tool for the treatment of cancers, including lung metastasis. Clin Cancer Res; 19(3); 668–79. ©2012 AACR.

Background While vaccine-induced antibodies are known to confer protection against incident human papillomavirus (HPV) infection, there is inconsistent data regarding the protective effect of naturally acquired anti-HPV antibodies. Objectives To estimate the protective effect of naturally acquired anti-HPV16 serum antibodies against incident anogenital infection with HPV16 in females aged 20–64 years and to assess whether antibodies influence the persistence/clearance of anogenital HPV16 infection. Study design 4432 women attending the organized national cervical cancer screening program in Slovenia were initially enrolled. 2199 and 1848 women had valid HPV DNA results obtained using PCR-based assays and HPV antibody serotyping results obtained using pseudovirion-based serological assay, at baseline and at three-year follow-up, respectively. Results Baseline HPV16 seroprevalence was 2.4-fold higher among HPV16 DNA-positive women (55.7% vs. 23.2%; p 0.01). Baseline HPV16 DNA-positive/seronegative women frequently acquired anti-HPV16 antibodies during follow-up (OR=8.2; 95% CI: 3.8–17.8). Baseline anti-HPV16 antibodies persisted at follow-up, irrespective of baseline HPV16 DNA status (OR=40.6; 95% CI: 30.3–54.5). Baseline HPV16 DNA-negative/seropositive women were less likely to acquire HPV16 infection at follow-up (unadjusted OR=0.2; 0.1–0.9). However, the age-adjusted association was non-significant (adjusted OR=0.3; 0.1–1.2). The tendency for protective effect was stronger among women older than 25 years (OR=0.2; 0.03–1.8). Baseline anti-HPV16 antibodies were not associated with persistence/clearance of HPV16 infection at follow-up (OR=0.8; 0.3–1.9). Conclusions Naturally acquired anti-HPV16 serum antibodies appeared to protect against anogenital HPV16 infection, but this association was at least partially confounded by age. Baseline anti-HPV16 serum antibodies did not influence persistence/clearance of HPV16 infection at follow-up.

Human papillomaviruses (HPV) cause cervical cancer and have recently also been implicated in mouth, laryngeal and anogenital cancers. There are three commercially available prophylactic vaccines that show good efficacy; however, efforts to develop second-generation vaccines that are more affordable, stable and elicit a wider spectrum of cross-neutralising immunity are still ongoing. Testing antisera elicited by current and candidate HPV vaccines for neutralizing antibodies is done using a HPV pseudovirion (PsV)-based neutralisation assay (PBNA). PsVs are produced by transfection of mammalian cell cultures with plasmids expressing L1 and L2 capsid proteins and a reporter gene plasmid, a highly expensive process. We investigated making HPV-16 PsVs in plants, in order to develop a cheaper alternative. The secreted embryonic alkaline phosphatase (SEAP) reporter gene and promoter were cloned into a geminivirus-derived plant expression vector, in order to produce circular dsDNA replicons. This was co-introduced into Nicotiana benthamiana plants with vectors expressing L1 and L2 via agroinfiltration and presumptive PsVs were purified. The PsVs contained DNA and could be successfully used for PBNA with anti-HPV antibodies. This is the first demonstration of the production of mammalian pseudovirions in plants and the first demonstration of the potential of plants to make DNA vaccines.

Identification of host factors that are needed for Zaire Ebolavirus (EBOV) entry provides insights into the mechanism(s) of filovirus uptake, and these factors may serve as potential antiviral targets. In order to identify novel host genes and pathways involved in EBOV entry, gene array findings in the National Cancer Institute's NCI-60 panel of human tumor cell lines were correlated with permissivity for EBOV glycoprotein (GP)-mediated entry. We found that the gene encoding the γ2 subunit of AMP-activated protein kinase (AMPK) strongly correlated with EBOV transduction in the tumor panel. The AMPK inhibitor compound C inhibited infectious EBOV replication in Vero cells and diminished EBOV GP-dependent, but not Lassa fever virus GPC-dependent, entry into a variety of cell lines in a dose-dependent manner. Compound C also prevented EBOV GP-mediated infection of primary human macrophages, a major target of filoviral replication in vivo . Consistent with a role for AMPK in filovirus entry, time-of-addition studies demonstrated that compound C abrogated infection when it was added at early time points but became progressively less effective when added later. Compound C prevented EBOV pseudovirion internalization at 37°C as cell-bound particles remained susceptible to trypsin digestion in the presence of the inhibitor but not in its absence. Mouse embryonic fibroblasts lacking the AMPKα1 and AMPKα2 catalytic subunits were significantly less permissive to EBOV GP-mediated infection than their wild-type counterparts, likely due to decreased macropinocytic uptake. In total, these findings implicate AMPK in macropinocytic events needed for EBOV GP-dependent entry and identify a novel cellular target for new filoviral antivirals.

Despite advancements in treatments, cancer remains a life-threatening disease that is resistant to therapy. Single-modal cancer therapy is often insufficient to provide complete remission. A revolution in cancer therapy may someday be provided by vector-based gene and drug delivery systems. However, it remains difficult to achieve this aim because viral and non-viral vectors have their own advantages and limitations. To overcome these limitations, virosomes have been constructed by combining viral components with non-viral vectors or by using pseudovirions without viral genome replication. Viruses, such as influenza virus, HVJ (hemagglutinating virus of Japan; Sendai virus) and hepatitis B virus, have been used in the construction of virosomes. The HVJ-derived vector is particularly promising due to its highly efficient delivery of DNA, siRNA, proteins and anti-cancer drugs. Furthermore, the HVJ envelope (HVJ-E) vector has intrinsic anti-tumor activities including the activation of multiple anti-tumor immunities and the induction of cancer-selective apoptosis. HVJ-E is currently being clinically used for the treatment of melanoma. A promising multi-modal cancer therapy will be achieved when virosomes with intrinsic anti-tumor activities are utilized as vectors for the delivery of anti-tumor drugs and genes.

Puumala (PUUV) and Hantaan (HTNV) viruses are hantaviruses within the family Bunyaviridae and associated with Hemorrhagic Fever with Renal Syndrome (HFRS) in humans. Little is known about how these viruses interact with host cells, though pathogenic hantaviruses interact with α v β 3 integrin. To study host cell interactions and rapidly test the ability of antibodies to prevent infection, we produced HTNV and PUUV pseudovirions on a vesicular stomatitis virus (VSV) core. Similar to replication-competent hantaviruses, infection was low-pH-dependent. Despite broad cell tropism, several human T cell lines were poorly permissive to hantavirus pseudovirions, compared to VSV, indicating a relative block to infection at the level of entry. Stable expression of α v β 3 integrin in SupT1 cells did not restore infectivity. Finally, the pseudovirion system provided a rapid, quantitative, and specific method to screen for neutralizing antibodies in immune sera.

The available virus-like particle (VLP)-based prophylactic vaccines against specific human papillomavirus (HPV) types afford close to 100% protection against the type-associated lesions and disease. Based on papillomavirus animal models, it is likely that protection against genital lesions in humans is mediated by HPV type-restricted neutralizing antibodies that transudate or exudate at the sites of genital infection. However, a correlate of protection was not established in the clinical trials because few disease cases occurred, and true incident infection could not be reliably distinguished from the emergence or reactivation of prevalent infection. In addition, the current assays for measuring vaccine-induced antibodies, even the gold standard HPV pseudovirion (PsV) in vitro neutralization assay, may not be sensitive enough to measure the minimum level of antibodies needed for protection. Here, we characterize the recently developed model of genital challenge with HPV PsV and determine the minimal amounts of VLP-induced neutralizing antibodies that can afford protection from genital infection in vivo after transfer into recipient mice. Our data show that serum antibody levels >100-fold lower than those detectable by in vitro PsV neutralization assays are sufficient to confer protection against an HPV PsV genital infection in this model. The results clearly demonstrate that, remarkably, the in vivo assay is substantially more sensitive than in vitro PsV neutralization and thus may be better suited for studies to establish correlates of protection.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged as the causal agent of an endemic atypical pneumonia, infecting thousands of people worldwide. Although a number of promising potential vaccines and therapeutic agents for SARS-CoV have been described, no effective antiviral drug against SARS-CoV is currently available. The intricate, sequential nature of the viral entry process provides multiple valid targets for drug development. Here, we describe a rapid and safe cell-based high-throughput screening system, Dual Envelope Pseudovirion (DEP) Assay, for specifically screening inhibitors of viral entry. The assay system employs a novel dual envelope strategy, using lentiviral pseudovirions as targets whose entry is driven by the SARS-CoV Spike glycoprotein. A second, unrelated viral envelope is used as an internal control to reduce the number of false positives. As an example of the power of this assay a class of inhibitors is reported with the potential to inhibit SARS-CoV at two steps of the replication cycle, viral entry and particle assembly. This assay system can be easily adapted to screen entry inhibitors against other viruses with the careful selection of matching partner virus envelopes.

Cellular entry of Ebola virus (EBOV), a deadly hemorrhagic fever virus, is mediated by the viral glycoprotein (GP). The receptor-binding subunit of GP must be cleaved (by endosomal cathepsins) in order for entry and infection to proceed. Cleavage appears to proceed through 50-kDa and 20-kDa intermediates, ultimately generating a key 19-kDa core. How 19-kDa GP is subsequently triggered to bind membranes and induce fusion remains a mystery. Here we show that 50-kDa GP cannot be triggered to bind to liposomes in response to elevated temperature but that 20-kDa and 19-kDa GP can. Importantly, 19-kDa GP can be triggered at temperatures ∼10°C lower than 20-kDa GP, suggesting that it is the most fusion ready form. Triggering by heat (or urea) occurs only at pH 5, not pH 7.5, and involves the fusion loop, as a fusion loop mutant is defective in liposome binding. We further show that mild reduction (preferentially at low pH) triggers 19-kDa GP to bind to liposomes, with the wild-type protein being triggered to a greater extent than the fusion loop mutant. Moreover, mild reduction inactivates pseudovirion infection, suggesting that reduction can also trigger 19-kDa GP on virus particles. Our results support the hypothesis that priming of EBOV GP, specifically to the 19-kDa core, potentiates GP to undergo subsequent fusion-relevant conformational changes. Our findings also indicate that low pH and an additional endosomal factor (possibly reduction or possibly a process mimicked by reduction) act as fusion triggers.

In a bioinformatics-based screen for cellular genes that enhance Zaire ebolavirus (ZEBOV) transduction, AXL mRNA expression strongly correlated with ZEBOV infection. A series of cell lines and primary cells were identified that require Axl for optimal ZEBOV entry. Using one of these cell lines, we identified ZEBOV entry events that are Axl-dependent. Interactions between ZEBOV-GP and the Axl ectodomain were not detected in immunoprecipitations and reduction of surface-expressed Axl by RNAi did not alter ZEBOV-GP binding, providing evidence that Axl does not serve as a receptor for the virus. However, RNAi knock down of Axl reduced ZEBOV pseudovirion internalization and α-Axl antisera inhibited pseudovirion fusion with cellular membranes. Consistent with the importance of Axl for ZEBOV transduction, Axl transiently co-localized on the surface of cells with ZEBOV virus particles and was internalized during virion transduction. In total, these findings indicate that endosomal uptake of filoviruses is facilitated by Axl.

While neutralizing antibodies (Nabs) mediate protection in humans against a diversity of viral pathogens (38, 53, 72), it is unclear how they impact human immunodeficiency virus type 1 (HIV-1) infection. One reason that the contribution of neutralizing antibodies to the control of HIV-1 remains uncertain is that HIV-specific neutralizing antibodies develop relatively late in natural infection. High titers of HIV-specific autologous neutralizing antibodies usually emerge as late as 2 to 3 months after infection and continue to evolve throughout the first years of infection (18, 25, 57, 66, 74). Such neutralizing antibodies have been shown to influence HIV-1 evolution within a host and to be responsible for viral escape mutations (47, 48, 58, 59). A better understanding of why there is a prolonged time associated with the maturation of the neutralizing antibody response in HIV-1 infection and whether conserved viral epitopes exist that could mediate antibody protection is important for the development of effective HIV-1 vaccine strategies. The simian immunodeficiency virus (SIV)/rhesus macaque model of AIDS provides an important system to study AIDS immunopathogenesis and to evaluate HIV-1 vaccine strategies. SIVmac251, an uncloned, pathogenic, CCR5-tropic virus isolate comprised of a swarm of quasispecies that are closely related (33), and SIVmac239, an infectious molecular clone derived from SIVmac251, are the two most commonly used rhesus monkey SIV challenge viruses utilized in AIDS vaccine research in the nonhuman primate (NHP) model. SIVmac239 has been shown to be relatively resistant to antibody-mediated neutralization by both autologous antibodies and a wide range of monoclonal antibodies (29, 30). The env sequence evolution in SIVmac239-infected rhesus monkeys and SIVMne-CL8-infected pigtailed macaques has been well described (8, 50, 51). Some of these changes in Env have been shown to result in viral escape from neutralizing antibodies (7, 10, 34, 60). In particular, a recent study by Sato et al. characterized SIVmac239 env sequence changes that were associated with viral escape in a rhesus monkey with an unusually high titer of neutralizing antibodies after intravenous infection (67). However, the antibody-mediated neutralization of SIVmac251 has not been tested rigorously using standardized assays that are currently being used to measure neutralization of HIV-1, thereby precluding a direct comparison of the neutralization sensitivities of HIV-1 and SIV. Furthermore, it is also unclear whether more typical titers of neutralizing antibodies against SIV239/251 exert selection pressure on the viral population in animals that acquire infection mucosally. The aims of this study were to elucidate the kinetics of the neutralizing antibody response against the transmitted viruses and the sequence evolution of env in association with humoral immunity in mucosally infected rhesus macaques. We hypothesized that a low titer of SIVmac Env-specific neutralizing antibodies exerts potent selection pressure on the viral quasispecies. To test this hypothesis, we utilized a pseudovirion-based TZM-bl reporter gene neutralization assay and single genome amplification (SGA) in order to characterize the humoral immune pressures driving viral sequence evolution in four rhesus monkeys that were infected with SIVmac251 via intrarectal inoculations.

This study developed and validated a high-throughput human papillomavirus (HPV) serology method based on Luminex technology, using pseudovirions (PsVs) of eight mucosal HPV types (HPV-6, -11, -16, -18, -31, -45, -52 and -58) and two cutaneous HPV types (HPV-5 and -38) bound to heparin-coated beads. Analysis with neutralizing type-specific monoclonal antibodies against the included HPV types indicated the type specificity of the assay. Analysis of negative-control serum samples from 63 children and 71 middle-aged women with up to one lifetime sexual partner indicated high specificity. Positive-control serum samples from subjects with known HPV DNA status or clinical diagnosis found expected sensitivities for most of the HPV types in 219 European serum samples, but lower than expected in 124 samples from Africa. HPV-45 and -52 did not react as expected with the human serum samples. The PsV-Luminex method was used to determine the HPV-seropositivity-associated relative risk for future cervical cancer using 208 serum samples from a prospective study of 18 814 women followed for 23 years, analysed previously with standard HPV-16 ELISA. The PsV-Luminex method gave similar results to ELISA (kappa=0.77). As expected, HPV seropositivities assayed using the PsV-Luminex method found an increased risk of cervical cancer for HPV-16 [odds ratio (OR)=7.7, 95 % confidence interval (CI)=2.6-23] and HPV-31 (OR=4.1, 95 % CI=1.6-10.8), non-significant tendencies for increased risk for other mucosal HPV types and no risk for the cutaneous HPV types. In summary, multiplexed HPV serology using mammalian-derived PsVs selected for native conformation by binding to heparin-coated beads was validated as a high-throughput HPV serological method for most of the analysed HPV types.

Functional studies of HIV-1 envelope glycoproteins (Envs) commonly include the generation of pseudoviruses, which are produced by co-transfection of rev–vpu–env cassettes with an env-deficient provirus. Here, we describe six Env constructs from transmitted/founder HIV-1 that were defective in the pseudotyping assay, although two produced infectious virions when expressed from their cognate proviruses. All of these constructs exhibited an unusual gene arrangement in which the first exon of rev (rev1) and vpu were in the same reading frame without an intervening stop codon. Disruption of the rev1–vpu fusion gene by frameshift mutation, stop codon, or abrogation of the rev initiation codon restored pseudovirion infectivity. Introduction of the fusion gene into wildtype Env cassettes severely compromised their function. The defect was not due to altered env and rev transcription or a dominant negative effect of the expressed fusion protein, but seemed to be caused by inefficient translation at the env initiation codon. Although the rev1–vpu polymorphism affects Env expression only in vitro, it can cause problems in studies requiring Env complementation, such as analyses of co-receptor usage and neutralization properties, since 3% of subtype A, 20% of subtype C and 5% of CRF01_A/E viruses encode the fusion gene. A solution is to eliminate the rev initiation codon when amplifying rev–vpu–env cassettes since this increases Env expression irrespective of the presence of the polymorphism.

The evolution of envelope mutations by replicating primate immunodeficiency viruses allows these viruses to escape from the immune pressure mediated by neutralizing antibodies. Vaccine-induced anti-envelope antibody responses may accelerate and/or alter the specificity of the antibodies, thus shaping the evolution of envelope mutations in the replicating virus. To explore this possibility, we studied the neutralizing antibody response and the envelope sequences in rhesus monkeys vaccinated with either gag - pol - nef immunogens or gag - pol - nef immunogens in combination with env and then infected with simian immunodeficiency virus (SIV). Using a pseudovirion neutralization assay, we demonstrate that envelope vaccination primed for an accelerated neutralizing antibody response following virus challenge. To monitor viral envelope evolution in these two cohorts of monkeys, full-length envelopes from plasma virus isolated at weeks 37 and 62 postchallenge were sequenced by single genome amplification to identify sites of envelope mutations. We show that env vaccination was associated with a change in the pattern of envelope mutations. Prevalent mutations in sequences from gag - pol - nef vaccinees included deletions in both variable regions 1 and 4 (V1 and V4), whereas deletions in the env vaccinees occurred only in V1. These data show that env vaccination altered the focus of the antibody-mediated selection pressure on the evolution of envelope following SIV challenge.

Over the last two decades, small interfering RNA (siRNA)-mediated gene silencing has quickly become one of the most powerful techniques used to study gene function in vitro and a promising area for new therapeutics. Delivery remains a significant impediment to realizing the therapeutic potential of siRNA, a problem that is also tied to immunogenicity and toxicity. Numerous delivery vehicles have been developed, including some that can be categorized as pseudovirions: these are vectors that are directly derived from viruses but whose viral coding sequences have been eliminated, preventing their classification as viral vectors. Characteristics of the pseudovirions discussed in this review, namely phagemids, HSV amplicons, SV40 in vitro-packaged vectors, influenza virosomes, and HVJ-Envelope vectors, make them attractive for the delivery of siRNA-based therapeutics. Pseudovirions were shown to deliver siRNA effector molecules and bring about RNA interference (RNAi) in various cell types in vitro, and in vivo using immune-deficient and immune-competent mouse models. Levels of silencing were not always determined directly, but the duration of siRNA-induced knockdown lasted at least 3 days. We present examples of the use of pseudovirions for the delivery of synthetic siRNA as well as the delivery and expression of DNA-directed siRNA.

The deduced amino acid ( aa ) sequence of L2 of the newly sequenced HPV51 strain, isolated by Matsukura and Sugase (Ma-strain), was markedly different from that of the prototype HPV51 isolated by Nuovo et al. (Nu-strain) (GenBank M62877 ) in two regions: aa 95–99 (region I) and aa 179–186 (region II). The two regions of Ma-strain were homologous to those of the other mucosal HPVs. The aa sequences of the N-terminal and C-terminal regions of Ma-L2 and Nu-L2 were identical and contained the nuclear localizing signal (NLS). When expressed in HEK293 cells, Ma-strain L2 (Ma-L2) was located in the nucleus but Nu-strain L2 (Nu-L2), in the cytoplasm. The chimeric L2s having both Nu-L2 regions I and II were located in the cytoplasm, and those having one of them were located both in the nucleus and cytoplasm, suggesting that Nu-L2 regions I and II inhibit the NLS function. For a better understanding of a role of L2 in infection, pseudovirion (PV) preparations were produced with a reporter, Ma-strain L1, and various L2s (Ma-L2, Nu-L2, or the chimeric L2s). These PV preparations contained structurally similar particles composed of L1 and L2 and the packaged reporter plasmid at a similar level. The reporter expression was not induced in HEK293 cells after inoculation with PVs containing the L2s that are incapable of localizing in the nucleus when expressed alone. Among PVs containing L2s capable of localizing in the nucleus, the reporter expression was induced only by PVs containing Ma-L2 region I. Thus, the results indicate that the expression of the reporter in the HPV51 PV requires the nuclear localizing ability of L2 and another unknown function associated with region I.