Interactions of HIV-1 Capsid with Host Factors and Their Implications for Developing Novel Therapeutics.

The Human Immunodeficiency Virus type 1 (HIV-1) virion contains a conical shell, termed capsid, encasing the viral RNA genome. After cellular entry of the virion, the capsid is released and ensures the protection and delivery of the HIV-1 genome to the host nucleus for integration. The capsid relies on many virus-host factor interactions which are regulated spatiotemporally throughout the course of infection. In this paper, we will review the current understanding of the highly dynamic HIV-1 capsid-host interplay during the early stages of viral replication, namely intracellular capsid trafficking after viral fusion, nuclear import, uncoating, and integration of the viral genome into host chromatin. Conventional anti-retroviral therapies primarily target HIV-1 enzymes. Insights of capsid structure have resulted in a first-in-class, long-acting capsid-targeting inhibitor, GS-6207 (Lenacapavir). This inhibitor binds at the interface between capsid protein subunits, a site known to bind host factors, interferes with capsid nuclear import, HIV particle assembly, and ordered assembly. Our review will highlight capsid structure, the host factors that interact with capsid, and high-throughput screening techniques, specifically genomic and proteomic approaches, that have been and can be used to identify host factors that interact with capsid. Better structural and mechanistic insights into the capsid-host factor interactions will significantly inform the understanding of HIV-1 pathogenesis and the development of capsid-centric antiretroviral therapeutics.

A Reduced Dose Whole Virion Aluminum Adjuvanted Seasonal Influenza Vaccine Is Immunogenic, Safe, and Well Tolerated in Pediatric Patients.

BACKGROUND: Data suggest that pediatric patients might react differently to influenza vaccination, both in terms of immunity and side effects. We have recently shown that using a whole virion vaccine with aluminum phosphate adjuvants, reduced dose vaccines containing 6 µg of viral hemagglutinin (HA) per strain are immunogenic, and well tolerated in adult and elderly patients. Here we show the results of a multicenter clinical trial of pediatric patients, using reduced doses of a new, whole virion, aluminum phosphate adjuvanted vaccine (FluArt, Budapest, Hungary). METHODS: A total of 120 healthy volunteers were included in two age groups (3-11 years, receiving 3 µg of HA per strain, and 12-18 years, receiving 6 µg of HA per strain). We used hemagglutination inhibition testing to assess immunogenicity, based on EMA and FDA licensing criteria, including post/pre-vaccination geometric mean titer ratios, seroconversion and seropositivity rates. Safety and tolerability were assessed using CHMP guidelines. RESULTS: All subjects entered the study and were vaccinated (ITT population). All 120 subjects attended the control visit on Day 21 (PP population). All immunogenicity licensing criteria were met in both age groups for all three vaccine virus strains. No serious adverse events were detected and the vaccine was well tolerated by both age groups. DISCUSSION: Using a whole virion vaccine and aluminum phosphate adjuvants, a reduction in the amount of the viral hemmaglutinin is possible while maintaining immunogenicity, safety and tolerability in pediatric and adolescent patients.

Future perspective: high-throughput construction of new ultrasensitive cytokine and virion liquid chips for high-throughput screening (HTS) of anti-inflammatory drugs or clinical diagnosis and treatment of inflammatory diseases.

Pathogen-host cell interactions play an important role in many human infectious and inflammatory diseases. Several pathogens, including Escherichia coli (E. coli), Mycobacterium tuberculosis (M. tb), and even the recent 2019 novel coronavirus (2019-nCoV), can cause serious breathing and brain disorders, tissue injury and inflammation, leading to high rates of mortality and resulting in great loss to human physical and mental health as well as the global economy. These infectious diseases exploit the microbial and host factors to induce serious inflammatory and immunological symptoms. Thus the development of anti-inflammatory drugs targeting bacterial/viral infection is an urgent need. In previous studies, YojI-IFNAR2, YojI-IL10RA, YojI-NRP1,YojI-SIGLEC7, and YojI-MC4R membrane-protein interactions were found to mediate E. coli invasion of the blood-brain barrier (BBB), which activated the downstream anti-inflammatory proteins NACHT, LRR and PYD domains-containing protein 2(NLRP2), using a proteomic chip conjugated with cell immunofluorescence labeling. However, the studies of pathogen (bacteria/virus)-host cell interactions mediated by membrane protein interactions did not extend their principles to broad biomedical applications such as 2019-nCoV infectious disease therapy. The first part of this feature article presents in-depth analysis of the cross-talk of cellular anti-inflammatory transduction signaling among interferon membrane protein receptor II (IFNAR2), interleukin-10 receptor subunit alpha (IL-10RA), NLRP2 and [Ca2+]-dependent phospholipase A2 (PLA2G5), based on experimental results and important published studies, which lays a theoretical foundation for the high-throughput construction of the cytokine and virion solution chip. The paper then moves on to the construction of the novel GPCR recombinant herpes virion chip and virion nano-oscillators for profiling membrane protein functions, which drove the idea of constructing the new recombinant virion and cytokine liquid chips for HTS of leading drugs. Due to the different structural properties of GPCR, IFNAR2, ACE2 and Spike of 2019-nCoV, their ligands will either bind the extracellular domain of IFNAR2/ACE2/Spike or the specific loops of the GPCR on the envelope of the recombinant herpes virions to induce dynamic charge distribution changes that lead to the variable electron transition for detection. Taken together, the combined overview of two of the most innovative and exciting developments in the immunoinflammatory field provides new insight into high-throughput construction of ultrasensitive cytokine and virion liquid chips for HTS of anti-inflammatory drugs or clinical diagnosis and treatment of inflammatory diseases including infectious diseases, acute or chronic inflammation (acute gouty arthritis or rheumatoid arthritis), cardiovascular disease, atheromatosis, diabetes, obesity, tissue injury and tumors. It has significant value in the prevention and treatment of these serious and painful diseases. Graphical abstract.

Immunocapture of virions with virus-specific antibodies prior to high-throughput sequencing effectively enriches for virus-specific sequences.

Virus discovery based on high-throughput sequencing relies on enrichment for virus sequences prior to library preparation to achieve a sufficient number of viral reads. In general, preparations of double-stranded RNA or total RNA preparations treated to remove rRNA are used for sequence enrichment. We used virus-specific antibodies to immunocapture virions from plant sap to conduct cDNA synthesis, followed by library preparation and HTS. For the four potato viruses PLRV, PVY, PVA and PYV, template preparation by virion immunocapture provided a simpler and less expensive method than the enrichment of total RNA by ribosomal depletion. Specific enrichment of viral sequences without an intermediate amplification step was achieved, and this high coverage of sequences across the viral genomes was important to identify rare sequence variations. Using this approach, the first complete genome sequence of a potato yellowing virus isolate (PYV, DSMZ PV-0706) was determined in this study. PYV can be confidently assigned as a distinct species in the genus Ilarvirus.

Chimeric Virus as a Source of the Potato Leafroll Virus Antigen.

Large quantities of potato leafroll virus (PLRV) antigen are difficult to obtain because this virus accumulates in plants at a low titer. To overcome this problem, we constructed a binary vector containing chimeric cDNA, in which the coat protein (CP) gene of the crucifer infecting tobacco mosaic virus (crTMV) was substituted for the coat protein gene of PLRV. The PLRV movement protein (MP) gene, which overlaps completely with the CP gene, was doubly mutated to eliminate priming of the PLRV MP translation from ATG codons with no changes to the amino acid sequence of the CP. The untranslated long intergenic region located upstream of the CP gene was removed from the construct. Transcribed powerful tobamovirus polymerase of the produced vector synthesized PLRV CP gene that was, in turn, translated into the protein. CP PLRV packed RNAs from the helical crTMV in spherical virions. Morphology, size and antigenic specificities of the wild-type and chimeric virus were similar. The yield of isolated chimera was about three orders higher than the yield of native PLRV. The genetic manipulations facilitated the generation of antibodies against the chimeric virus, which recognize the wild-type PLRV.

Antibody and cell-mediated immune response to whole virion and split virion influenza vaccine in patients with inflammatory bowel disease on maintenance immunosuppressive and biological therapy.

OBJECTIVE: Influenza vaccination is recommended for inflammatory bowel disease (IBD) patients on immunosuppressive therapy. The objective was to evaluate the antibody and cell-mediated immune response to the split and whole virion influenza vaccine in patients with IBD treated with anti-TNF-α and immunosuppressive therapy. PATIENTS AND METHODS: One hundred and fifty-six immunocompromised IBD patients were vaccinated. Fifty-three patients (control group) refused vaccination. Split virion vaccine and whole virion vaccine were used. Serum samples were obtained for pre- and postimmunization antibody titers to influenza vaccine (A/California/7/2009 [H1N1], A/Victoria/361/2011 [H3N2], B/Wisconsin/1/2010-like B/Hubei-Wujiagang/158/2009). Cell-mediated response was evaluated using an interferon (INF)-γ, interleukine (IL)-2 and tumor necrosis factor (TNF)-α ELISA. RESULTS: Postimmunization titers of both influenza subtypes increased significantly after the administration of split virion vaccines compared to the controls and to those who received whole virion vaccine. The antibody titers of Influenza B also increased significantly in patients immunized with split vaccine and treated with anti-TNF-α therapy. After influenza vaccination, the level of serum IL-2 significantly decreased. No serious side effects developed occurred after influenza vaccination, and the influenza-like symptoms did not differ significantly between vaccinated versus control patients. The relapse of the disease was observed in only 10% of the patients and was more common in vaccinated than in control subjects. CONCLUSION: Split virion vaccines seem to be more effective than whole virion vaccines. Measuring the antibody responses is worthwhile in patients treated with immunosuppressants to determine the efficacy of influenza vaccination.

Protective efficacy of baculovirus-derived influenza virus-like particles bearing H5 HA alone or in combination with M1 in chickens.

Since 2003, the highly pathogenic avian influenza (HPAI) H5N1 has become a serious problem in animals and an increasing threat to public health. To develop effective vaccines for H5 HPAI in chickens, virus-like particles (VLP) were produced using a baculovirus expression system. The particles comprised hemagglutinin (HA) alone (HA-VLP) or HA in combination with a matrix protein (M1; HAM-VLP) derived from a recent clade 2.3.2.1 H5N1 HPAI virus. To compare the immunogenicity and protective efficacy of these VLPs, 10 µg HAM-VLP, the equivalent amounts of HA incorporated HA-VLP or whole inactivated virus (WIV), were emulsified with mineral oil and used to immunize chickens. The serum hemagglutination inhibition antibody levels induced by HA-VLP and HAM-VLP were comparable to WIV. Antibodies to nucleoprotein were detected only in the WIV group. Immunized chickens in each group survived and were protected against a lethal homologous virus challenge, showing no clinical signs of infection. The challenge virus was detected intermittently in some oropharyngeal swabs, but not in cloacal swabs or various organs, which means that VLPs and WIV provide protection against systemic but not local virus replication in chickens. After the challenge, the HA-VLP group showed significantly increased serum antibody levels compared to the HAM-VLP and WIV groups, and some chickens in the HA-VLP group seroconverted with respect to nucleoprotein. Taken together, these results suggest that VLPs may be an effective method for controlling HPAI in chickens. They could be applied to a differentiating infected from vaccinated animals (DIVA) strategy. In addition, it is likely that HAM-VLP is more efficacious than HA-VLP in chickens.

A/H5N1 prepandemic influenza vaccine (whole virion, vero cell-derived, inactivated) [Vepacel®].

The influenza A subtype H5N1 virus is a likely causative agent for the next human influenza pandemic. Pandemic influenza vaccine production can begin only after a novel pandemic virus emerges. Cell-based vaccine production has advantages over conventional egg-based methods, allowing more rapid large-scale vaccine production. A reliable Vero cell culture system is available for pandemic and prepandemic influenza vaccine production. Prepandemic influenza vaccines are an important component of influenza pandemic preparedness plans, as their targeted use in the pandemic alert period or early in a pandemic is likely to mitigate the consequences of an influenza outbreak. Vepacel® is a prepandemic influenza vaccine (whole virion, Vero cell-derived, inactivated) containing antigen of H5N1 strain A/Vietnam/1203/2004 and is approved for use in the EU. Clinical immunogenicity studies with the vaccine have demonstrated good rates of functional neutralizing antibody responses against the vaccine strain (A/Vietnam/1203/2004), meeting established immunogenicity criteria for seasonal influenza vaccines, and cross-reactivity against H5N1 strains from other clades. In phase I/II and III studies, a heterologous (A/Indonesia/05/2005) booster vaccine administered to healthy adult and elderly volunteers 6-24 months after the two-dose priming vaccine (A/Vietnam/1203/2004) regimen induced good immunogenic responses against both H5N1 strains, demonstrating strong immunological memory. Broadly similar, albeit less robust, responses were observed in two special risk cohorts of immunocompromised and chronically ill patients. In general, adverse events observed in clinical immunogenicity studies with H5N1 vaccine (A/Vietnam/1203/2004) were similar to those reported with non-adjuvanted, inactivated, seasonal influenza vaccines.

Safety and immunogenicity of AS03B adjuvanted split virion versus non-adjuvanted whole virion H1N1 influenza vaccine in UK children aged 6 months-12 years: open label, randomised, parallel group, multicentre study.

OBJECTIVES: To compare the safety, reactogenicity, and immunogenicity of an adjuvanted split virion H1N1 vaccine and a non-adjuvanted whole virion vaccine used in the pandemic immunisation programme in the United Kingdom. DESIGN: Open label, randomised, parallel group, phase II study. SETTING: Five UK centres (Oxford, Southampton, Bristol, Exeter, and London). PARTICIPANTS: Children aged 6 months to less than 13 years for whom a parent or guardian had provided written informed consent and who were able to comply with study procedures were eligible. Those with laboratory confirmed pandemic H1N1 influenza or clinically diagnosed disease meriting antiviral treatment, allergy to egg or any other vaccine components, or coagulation defects, or who were severely immunocompromised or had recently received blood products were excluded. Children were grouped by age: 6 months-<3 years (younger group) and 3-<13 years (older group). Recruitment was by media advertising and direct mailing. Recruitment visits were attended by 949 participants, of whom 943 were enrolled and 937 included in the per protocol analysis. INTERVENTIONS: Participants were randomised 1:1 to receive AS03(B) (tocopherol based oil in water emulsion) adjuvanted split virion vaccine derived from egg culture or non-adjuvanted whole virion vaccine derived from cell culture. Both were given as two doses 21 days apart. Reactogenicity data were collected for one week after immunisation by diary card. Serum samples were collected at baseline and after the second dose. MAIN OUTCOME MEASURES: Primary reactogenicity end points were frequency and severity of fever, tenderness, swelling, and erythema after vaccination. Immunogenicity was measured by microneutralisation and haemagglutination inhibition assays. The primary immunogenicity objective was a comparison between vaccines of the percentage of participants showing seroconversion by the microneutralisation assay (fourfold rise to a titre of >or=1:40 from before vaccination to three weeks after the second dose). RESULTS: Seroconversion rates were higher after the adjuvanted split virion vaccine than after the whole virion vaccine, most notably in the youngest children (163 of 166 participants with paired serum samples (98.2%, 95% confidence interval 94.8% to 99.6%) v 157 of 196 (80.1%, 73.8% to 85.5%), P<0.001) in children under 3 years and 226 of 228 (99.1%, 96.9% to 99.9%) v 95.9%, 92.4% to 98.1%, P=0.03) in those over 3 years). The adjuvanted split virion vaccine was more reactogenic than the whole virion vaccine, with more frequent systemic reactions and severe local reactions in children aged over 5 years after dose one (13 (7.2%, 3.9% to 12%) v 2 (1.1%, 0.1% to 3.9%), P<0.001) and dose two (15 (8.5%, 4.8% to 13.7%) v 2 (1.1%, 0.1% to 4.1%), P<0.002) and after dose two in those under 5 years (15 (5.9%, 3.3% to 9.6%) v 0 (0.0%, 0% to 1.4%), P<0.001). Dose two of the adjuvanted split virion vaccine was more reactogenic than dose one, especially for fever >or=38 masculineC in those aged under 5 (24 (8.9%, 5.8% to 12.9%) v 57 (22.4%, 17.5% to 28.1%), P<0.001). CONCLUSIONS: In this first direct comparison of an AS03(B) adjuvanted split virion versus whole virion non-adjuvanted H1N1 vaccine, the adjuvanted vaccine, while more reactogenic, was more immunogenic and, importantly, achieved high seroconversion rates in children aged less than 3 years. This indicates the potential for improved immunogenicity of influenza vaccines in this age group. TRIAL REGISTRATION: Clinical trials.gov NCT00980850; ISRCTN89141709.

Binding of Norovirus virus-like particles (VLPs) to human intestinal Caco-2 cells and the suppressive effect of pasteurized bovine colostrum on this VLP binding.

Noroviruses (NoVs), which cannot be grown in cell culture, are a major infectious agent of gastroenteritis. An in vitro assay system was established for the evaluation of NoV binding to enterocytes using virus-like particles (VLPs) produced in a baculovirus system expressing a NoV VP1 capsid protein. After confirmation of the purity by MS analysis, VLPs were incubated with human intestinal Caco-2 cells. NoV VLPs were detected clearly by confocal laser microscopy only on a certain population of Caco-2 cells, and were semi-quantified by immunoblotting of cell lysates. Then the suppressive effect of pasteurized bovine colostrum was analyzed on the VLP binding to Caco-2 cells by immunoblotting. The colostrum reduced VLP binding in a dose-dependent manner, at about 50% suppression with 12.5 microg of the colostral proteins. Furthermore, the colostrum contained IgG antibodies reacting to VLPs, suggesting that cross-reactive antibodies in the bovine colostrums block human NoV binding to intestinal cells.

A vaccine for hypertension based on virus-like particles: preclinical efficacy and phase I safety and immunogenicity.

BACKGROUND: Despite the availability of efficacious drugs, the success of treating hypertension is limited by patients' inconsistent drug intake. Immunization against angiotensin II may offer a valuable alternative to conventional drugs for the treatment of hypertension, because vaccines induce relatively long-lasting effects and do not require daily dosing. Here we describe the preclinical development and the phase I clinical trial testing of a virus-like particle (VLP)-based antihypertensive vaccine. METHODS AND RESULTS: An angiotensin II-derived peptide was conjugated to the VLP Qbeta (AngQb). AngQb was highly immunogenic in mice and rats. To test for efficacy, spontaneously hypertensive rats (SHR) were immunized with 400 microg AngQb or VLP alone. Group mean systolic blood pressure (SBP) was reduced by up to 21 mmHg (159 +/- 2 versus 180 +/- 5 mmHg, P < 0.001), and total angiotensin II levels (antibody-bound and free) were increased ninefold (85 +/- 20 versus 9 +/- 1 pmol/l, P = 0.002) compared with VLP controls. SHR treated with the angiotensin-converting enzyme (ACE) inhibitor ramipril (1 mg/kg per day by mouth) reached an SBP of 155 +/- 2 mmHg. Twelve healthy volunteers of a placebo-controlled randomized phase I trial were injected once with 100 microg AngQb. Angiotensin II-specific antibodies were raised in all subjects (100% responder rate) and AngQb was well tolerated. CONCLUSIONS: AngQb reduces blood pressure in SHR to levels obtained with an ACE inhibitor, and is immunogenic and well tolerated in humans. Therefore, vaccination against angiotensin II has the potential to become a useful antihypertensive treatment providing long-lasting effects and improving patient compliance.

Oral immunogenicity of human papillomavirus-like particles expressed in potato.

Human papillomavirus-like particles (HPV VLPs) have shown considerable promise as a parenteral vaccine for the prevention of cervical cancer and its precursor lesions. Parenteral vaccines are expensive to produce and deliver, however, and therefore are not optimal for use in resource-poor settings, where most cervical HPV disease occurs. Transgenic plants expressing recombinant vaccine immunogens offer an attractive and potentially inexpensive alternative to vaccination by injection. For example, edible plants can be grown locally and can be distributed easily without special training or equipment. To assess the feasibility of an HPV VLP-based edible vaccine, in this study we synthesized a plant codon-optimized version of the HPV type 11 (HPV11) L1 major capsid protein coding sequence and introduced it into tobacco and potato. We show that full-length L1 protein is expressed and localized in plant cell nuclei and that expression of L1 in plants is enhanced by removal of the carboxy-terminal nuclear localization signal sequence. We also show that plant-expressed L1 self-assembles into VLPs with immunological properties comparable to those of native HPV virions. Importantly, ingestion of transgenic L1 potato was associated with activation of an anti-VLP immune response in mice that was qualitatively similar to that induced by VLP parenteral administration, and this response was enhanced significantly by subsequent oral boosting with purified insect cell-derived VLPs. Thus, papillomavirus L1 protein can be expressed in transgenic plants to form immunologically functional VLPs, and ingestion of such material can activate potentially protective humoral immune responses.

Critical role for activation of antigen-presenting cells in priming of cytotoxic T cell responses after vaccination with virus-like particles.

Virus-like particles (VLPs) are known to induce strong Ab responses in the absence of adjuvants. In addition, VLPs are able to prime CTL responses in vivo. To study the efficiency of this latter process, we fused peptide p33 derived from lymphocytic choriomeningitis virus to the hepatitis B core Ag, which spontaneously assembles into VLPs (p33-VLPs). These p33-VLPs were efficiently processed in vitro and in vivo for MHC class I presentation. Nevertheless, p33-VLPs induced weak CTL responses that failed to mediate effective protection from viral challenge. However, if APCs were activated concomitantly in vivo using either anti-CD40 Abs or CpG oligonucleotides, the CTL responses induced were fully protective against infection with lymphocytic choriomeningitis virus or recombinant vaccinia virus. Moreover, these CTL responses were comparable to responses generally induced by live vaccines, because they could be measured in primary ex vivo (51)Cr release assays. Thus, while VLPs alone are inefficient at inducing CTL responses, they become very powerful vaccines if applied together with substances that activate APCs.

Lactogenic antibody responses in cows vaccinated with recombinant bovine rotavirus-like particles (VLPs) of two serotypes or inactivated bovine rotavirus vaccines.

Triple-layered virus-like particles (VLPs) were produced in a baculovirus expression system from the two prevalent bovine rotavirus (BRV) serotypes, IND (P[5]G6) and 2292B (P[11]G10). Five groups of pregnant cows were inoculated intramuscularly and intramammarily with IND VLPs [BRV RF VP2, and IND VP4, 6, and 7, 250 microg per dose], 2292B VLPs [RF VP2, Cr VP4 (P[11]), and 2292B VP6 and 7, 250 microg per dose], combined IND/2292B VLPs (125 microg each VLP per dose), inactivated IND BRV (5x10(7)PFU per dose, pre-inactivation), or cell supernatant (mock-controls) in incomplete Freund's adjuvant. Serum, colostrum and milk were collected and tested for isotype-specific antibodies, and homologous and heterologous neutralizing antibodies (VN) to BRV by ELISA and VN tests, respectively. After vaccination, the IgG1 and homologous VN geometric mean antibody titers (GMTs) to BRV in serum of vaccinated groups were significantly (P<0.05) higher than in the mock-controls through postpartum day (PPD) 30. In colostrum, the IgG1 and IgA, and the homologous and heterologous VN GMTs of the IND VLP, 2292B VLP, combined IND/2292B VLP and the inactivated IND groups were significantly enhanced compared to the mock-controls, except for the heterologous VN GMTs in the inactivated IND group. However, the VLP vaccine groups had significantly higher homologous and heterologous VN GMTs than the inactivated IND group. The VN GMTs of the IND/2292B VLP group were statistically similar to the homologous VN GMTs of the IND or 2292B VLP groups, although the IgG1 GMT was lower. In milk, the IgG1 and homologous VN GMTs of the VLP groups were significantly higher than the inactivated IND or the mock-control groups through PPD30. However, the heterologous and homologous VN GMTs of inactivated IND group were statistically similar to the mock-control group at PPD0 and 30, respectively. These results demonstrate that the BRV antibody titers in serum, colostrum and milk are significantly enhanced by the use of triple-layered VLPs and inactivated IND vaccines, but significantly higher antibody responses were observed in the VLP vaccinated cows. The combined IND/2292B VLP vaccine induced comparable VN responses to BRV in serum, colostrum and milk compared to those induced by the individual IND or 2292B VLP vaccines, suggesting that at least two different serotypes can be mixed to confer maximum antibody responses to the incorporated serotypes.

Developing HPV virus-like particle vaccines to prevent cervical cancer: a progress report.

BACKGROUND: the knowledge that sexually transmitted infection with one of a limited number of human papillomaviruses (HPVs) is a central cause of almost all cervical cancers affords the opportunity to prevent this common cancer through anti-viral vaccination. OBJECTIVE: the spectacular success of vaccines in preventing several other viral diseases offers hope that immunoprophylaxis against the relevant HPVs could lead to a major reduction in cervical cancer incidence. RESULTS AND CONCLUSION: the results of preclinical studies and early phase clinical trials of virus-like particle (VLP) based subunit vaccines have been very encouraging. However, unique aspects of papillomavirus biology and genital tract infections, and the lack of sexual a transmission model for papillomavirus, make it far from certain that effective prophylactic vaccination against genital HPV infection will be easily achieved. Future clinical efficacy trials will likely test the hypothesis that parenteral injection of VLPs can induce antibody mediated and type specific protection against genital tract HPV infection and subsequent development of premalignant neoplastic disease.

Isotype-specific antibody responses to rotavirus and virus proteins in cows inoculated with subunit vaccines composed of recombinant SA11 rotavirus core-like particles (CLP) or virus-like particles (VLP).

The isotype antibody responses to bovine IND P5, G6 and simian SA11 P2, G3 rotavirus and SA11 rotavirus proteins (VP4, VP6 and VP7) in serum, colostrum and milk were analysed by ELISA in three groups of vaccinated cows and nonvaccinated controls. Pregnant cows were vaccinated intramuscularly and intramammarily with recombinant baculovirus-expressed SA11 rotavirus VLP (triple-layered virus-like particles containing rotavirus VP2, VP4, VP6 and VP7); CLP (double-layered core-like particles containing rotavirus VP2 and VP6); or inactivated SA11 rotavirus, respectively. Rotavirus antigen titers were highest (30-200-fold) in ELISA in the VLP vaccine compared to the inactivated SA11 vaccine. The IgG1, IgG2 and IgM geometric mean antibody titers (GMT) to rotavirus (titers to bovine rotavirus vs SA11 rotavirus did not differ significantly for any isotype or group) and the IgG2 GMT to VP6 in serum at calving in the vaccinated groups were significantly (P < 0.05) higher than in the control group. In colostrum, IgG1 and IgA rotavirus antibody titers were significantly elevated for VLP (IgG1 GMT 832225; IgA GMT 16384), CLP (IgG1 GMT 660561; IgA GMT 10321) and SA11 (IgG1 GMT 131072; IgA GMT 1448) vaccinated cows compared to control cows (IgG1 GMT 11585; IgA GMT 45). The IgG1 and IgA GMT to rotavirus were significantly elevated (6-100-fold) in milk of VLP and CLP vaccinated cows compared to SA11 vaccinated or control cows. The isotype antibody responses to VP6 in serum, colostrum and milk paralleled the responses to rotavirus, but titers were approximately 2-10-fold lower. Only cows vaccinated with VLP had significantly enhanced serum, colostral and milk antibody titers to rotavirus VP4 and VP7. These results demonstrate that rotavirus antibody titers in serum, colostrum and milk are significantly enhanced by use of non-infectious VLP, CLP and inactivated SA11 rotavirus vaccines, but the VLP or CLP vaccines induced the highest antibody responses, corresponding to their higher rotavirus antigen titers measured by ELISA.

Construction, expression, and immunogenicity of chimeric HIV-1 virus-like particles.

The group-specific antigens Pr55gag of human immunodeficiency virus type-1 (HIV-1) self-assemble into noninfectious virus-like particles (VLP) that are released from various eucaryotic cells by budding. Deletion analysis of Pr55gag mutants revealed three domains into which sequences of the third variable domain V3 or the CD4-binding domain of the gp120 external glycoprotein can be inserted without destroying the capacity of the chimeric proteins to assemble to VLP. Immunization of rabbits with different types of purified chimeric VLP without adjuvants raised a strong antibody response to the Pr55gag carrier component. The magnitude of the antibody response to the inserted gp 120 epitopes strictly depended on their position within the gag polyprotein. These antisera exhibited only weak neutralizing activity. However, BALB/c mice immunized by different routes with different types of chimeric Pr55gag/V3 VLP without adjuvants developed a strong MHC class I (Dd)-restricted, cytolytic CD8+ T-cell (CTL) reactivity against a known epitope within the V3 domain. When the recombinant antigen was emulsified in mineral oil (incomplete Freund's adjuvant) or adsorbed in aluminium hydroxide, its immunogenicity for CTL was drastically reduced or completely abrogated. The magnitude of the V3-specific CTL response was not influenced by the position of the V3 domain within the Pr55gag-carrier moiety; the flanking residues, hence, did not influence processing of the exogenous antigen for MHC class I-restricted peptide presentation. These results indicate ways for the rational design and optimal delivery of CTL-stimulating HIV candidate vaccines.

All rabbits immunized with type A influenza virions have a serum haemagglutination-inhibition antibody response biased to a single epitope in antigenic site B.

Nine rabbits were immunized with type A influenza virions and the epitope specificities of the secondary serum haemagglutination-inhibition (HI) antibody response were analysed with a panel of neutralizing monoclonal (MAb) antibody double escape mutants. Each of the latter was made by sequential selection using a MAb directed to an epitope of a discrete antigenic site, site A, site B or site D, of the haemagglutinin (HA). Thus the epitope reactivity of the escape mutants was represented as A+ B- D-, A- B+ D- and A- B- D+. The HI antibody response of all antisera was biased to the site B epitope. In 9/12 antisera, obtained from seven rabbits immunized with whole virions, the site B epitope was predominant, representing 65-82% of the total HI antibody. The restriction of HI antibody was unaffected by strain of rabbit, route of inoculation (intravenous or subcutaneous), use of Freund's adjuvant, and up to four immunizing injections. In 3/7 rabbits immunized with whole virus, there was a HI antibody response to the HC2 (site A) or HC10 (site D) epitope, but not both, of equal magnitude to the site B epitope. The HI antibody response in one of the rabbits (#40) became more biased to the site B epitope between the third and fourth immunizing doses. Two further rabbits were immunized with virions which had been partially digested with bromelain and then purified from free HA. Both of these made equal HI antibody responses to the site B epitope and the site D epitope, possibly because their remaining HA spikes were better exposed. Overall, these data demonstrate an unexpected degree of restriction in the production of biologically relevant antibody, such that some rabbits (e.g. #45) mount an HI antibody response which is essentially epitope-specific. Implications for epitope specificity of HI antibody stimulated by human influenza vaccines, and also for the generation of antigenic drift variants are discussed. The reason for the non-responsiveness of the immune system to the many other HI epitopes of the HA is not known.

[A comparison of the protective properties of preparations of the virion and nonvirion ("soluble") antigens of the tick-borne encephalitis virus]. / Sopostavlenie protektivnykh svoistv preparatov virionnogo i nevirionnogo ("rastvorimogo") antigenov virusa kleshchevogo éntsefalita.

A comparative assessment of the protective properties of virion (VA) and nonvirion ("soluble") (NA) antigens of tick-borne encephalitis virus prepared as inactivated samples close in their parameters to vaccine preparations was carried out. The NA in the preparations free from VA or containing only trace, nonprotective amounts of it, was shown to have significantly lower protective properties than VA and exerted no booster effect on the protective activity when added to VA preparations.