Background

Infection with HPV 16 and 18, the major causative agents of cervical cancer, can be prevented through vaccination with a bivalent or quadrivalent vaccine. Both vaccines provide cross-protection against HPV-types not included in the vaccines. In particular, the bivalent vaccine provides additional protection against HPV 31, 33, and 45 and the quadrivalent vaccine against HPV31. The quadrivalent vaccine additionally protects against low-risk HPV type 6 and 11, responsible for most cases of genital warts. In this study, we made an analytical comparison of the two vaccines in terms of cost-effectiveness including the additional benefits of cross-protection and protection against genital warts in comparison with a screening-only strategy.

Methods

We used a Markov model, simulating the progression from HPV infection to cervical cancer or genital warts. The model was used to estimate the difference in future costs and health effects of both HPV-vaccines separately.

Results

In a cohort of 100,000 women, use of the bivalent or quadrivalent vaccine (both at 50% vaccination coverage) reduces the cervical cancer incidence by 221 and 207 cases, corresponding to ICERs of €17,600/QALY and €18,900/QALY, respectively. It was estimated that the quadrivalent vaccine additionally prevents 4390 cases of genital warts, reducing the ICER to €16,300/QALY. Assuming a comparable willingness to pay for cancer and genital warts prevention, the difference in ICERs could justify a slightly higher price (~7% per dose) in favor of the quadrivalent vaccine.

Conclusions

Clearly, HPV vaccination has been implemented for the prevention of cervical cancer. From this perspective, use of the bivalent HPV vaccine appears to be most effective and cost-effective. Including the benefits of prevention against genital warts, the ICER of the quadrivalent HPV vaccine was found to be slightly more favourable. However, current decision-making on the introduction of HPV is driven by the primary cervical cancer outcome. New vaccine tenders could consider the benefits of cross-protection and the benefits of genital warts, which requires more balanced decision-making.

Background. The decline in influenza vaccine efficacy in older adults is associated with a limited ability of current split-virus vaccines (SVVs) to stimulate cytotoxic T lymphocyte (CTL) responses required for clinical protection against influenza.

Methods. The Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant–stable emulsion (GLA-SE) was combined with SVV to stimulate peripheral blood mononuclear cells (PBMCs) in vitro to determine the cytokine response in dendritic cell subsets. Stimulated PBMCs were then challenged with live influenza virus to mimic the response to natural infection following vaccination, using previously identified T-cell correlates of protection.

Results. GLA-SE significantly increased the proportion of myeloid dendritic cells that produced tumor necrosis factor α, interleukin 6, and interleukin 12. When combined with SVV to stimulate PBMCs in vitro, this effect of GLA-SE was shown to regulate a T-helper 1 cell response upon challenge with live influenza virus; interleukin 10 production was suppressed, thus significantly increasing the interferon γ to interleukin 10 ratio and the cytolytic (granzyme B) response to influenza virus challenge, both of which have been shown to correlate with protection against influenza in older adults.

Conclusions. Our findings suggest that a novel adjuvant, GLA-SE, combined with standard SVV has the potential to significantly improve vaccine-mediated protection against influenza in older adults.

Identification of safe and effective adjuvants remains an urgent need for the development of inactivated influenza vaccines for mucosal administration. Here, we used a murine challenge model to evaluate the adjuvant activity of GPI-0100, a saponin-derived adjuvant, on influenza subunit vaccine administered via the intranasal or the intrapulmonary route. Balb/c mice were immunized with 1 µg A/PR/8 (H1N1) subunit antigen alone or in combination with varying doses of GPI-0100. The addition of GPI-0100 was required for induction of mucosal and systemic antibody responses to intranasally administered influenza vaccine and significantly enhanced the immunogenicity of vaccine administered via the intrapulmonary route. Remarkably, GPI-0100-adjuvanted influenza vaccine given at a low dose of 2×1 µg either in the nares or directly into the lungs provided complete protection against homologous influenza virus infection.

Background

The Dutch Human Papillomavirus (HPV) catch-up vaccination program in 2009 appeared less successful than expected. We aimed to identify the most important determinants of refusing the vaccination.

Methods

Two thousand parents of girls born in 1996 targeted for HPV vaccination received an invitation letter to participate in a questionnaire study. Two study groups were defined: the first group consisted of parents of girls who had accepted the vaccine and already received the first dose of HPV vaccination. The second group consisted of parents whose daughters were not vaccinated. The questionnaire consisted of a broad spectrum of possible determinants that were revealed after literature search and discussions with the stakeholders.

Results

Four hundred sixty nine questionnaires (24%) were returned, 307 (31%) from those who accepted and 162 (16%) from those who declined the vaccine. The decision not to accept the vaccine was largely determined by: (i) perception that the information provided by the government about the vaccine was limited or biased (OR 13.27); (ii) limited trust, that the government would stop the vaccination program if there were serious side effects (OR 9.95); (iii) lack of knowledge about the effectiveness of the vaccine (OR 7.67); (iv) concerns about the side effects of the vaccine (OR 4.94); (v) lack of conviction that HPV can be extremely harmful (OR 3.78); (vi) perception that the government is strongly influenced by vaccine producers (OR 3.54); and (vii) religious convictions (OR 2.18).

Conclusions

This study revealed several determinants for HPV vaccination uptake after implementation of the HPV vaccine for adolescent girls. These determinants should be taken into consideration in order to successfully implement HPV vaccination into National Immunization Programs.

Respiratory Syncytial Virus (RSV) is a major cause of viral brochiolitis in infants and young children and is also a significant problem in elderly and immuno-compromised adults. To date there is no efficacious and safe RSV vaccine, partially because of the outcome of a clinical trial in the 1960s with a formalin-inactivated RSV vaccine (FI-RSV). This vaccine caused enhanced respiratory disease upon exposure to the live virus, leading to increased morbidity and the death of two children. Subsequent analyses of this incident showed that FI-RSV induces a Th2-skewed immune response together with poorly neutralizing antibodies. As a new approach, we used reconstituted RSV viral envelopes, i.e. virosomes, with incorporated monophosphoryl lipid A (MPLA) adjuvant to enhance immunogenicity and to skew the immune response towards a Th1 phenotype. Incorporation of MPLA stimulated the overall immunogenicity of the virosomes compared to non-adjuvanted virosomes in mice. Intramuscular administration of the vaccine led to the induction of RSV-specific IgG2a levels similar to those induced by inoculation of the animals with live RSV. These antibodies were able to neutralize RSV in vitro. Furthermore, MPLA-adjuvanted RSV virosomes induced high amounts of IFNγ and low amounts of IL5 in both spleens and lungs of immunized and subsequently challenged animals, compared to levels of these cytokines in animals vaccinated with FI-RSV, indicating a Th1-skewed response. Mice vaccinated with RSV-MPLA virosomes were protected from live RSV challenge, clearing the inoculated virus without showing signs of lung pathology. Taken together, these data demonstrate that RSV-MPLA virosomes represent a safe and efficacious vaccine candidate which warrants further evaluation.

Flavivirus-infected cells secrete a mixture of mature, partially immature, and fully immature particles into the extracellular space. Although mature virions are highly infectious, prM-containing fully immature virions are noninfectious largely because the prM protein inhibits the cell attachment and fusogenic properties of the virus. If, however, cell attachment and entry are facilitated by anti-prM antibodies, immature flavivirus becomes infectious after efficient processing of the prM protein by the endosomal protease furin. A recent study demonstrated that E53, a cross-reactive monoclonal antibody (MAb) that engages the highly conserved fusion-loop peptide within the flavivirus envelope glycoprotein, preferentially binds to immature flavivirus particles. We investigated here the infectious potential of fully immature West Nile virus (WNV) and dengue virus (DENV) particles opsonized with E53 MAb and observed that, like anti-prM antibodies, this anti-E antibody also has the capacity to render fully immature flaviviruses infectious. E53-mediated enhancement of both immature WNV and DENV depended on efficient cell entry and the enzymatic activity of the endosomal furin. Furthermore, we also observed that E53-opsonized immature DENV particles but not WNV particles required a more acidic pH for efficient cleavage of prM by furin, adding greater complexity to the dynamics of antibody-mediated infection of immature flavivirus virions.

Cross-reactive dengue virus (DENV) antibodies directed against the envelope (E) and precursor membrane (prM) proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM antibodies render essentially non-infectious immature DENV infectious in Fcγ-receptor-expressing cells. Immature DENV particles are abundantly present in standard (st) virus preparations due to inefficient processing of prM to M during virus maturation. Structural analysis has revealed that the E protein is exposed in immature particles and this prompted us to investigate whether antibodies to E render immature particles infectious. To this end, we analyzed the enhancing properties of 27 anti-E antibodies directed against distinct structural domains. Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner. The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV) mouse model. Remarkably, mice injected with immature WNV opsonized with anti-E mAbs or immune serum produced a lethal infection in a dose-dependent manner, whereas in the absence of antibody immature WNV virions caused no morbidity or mortality. Furthermore, enhancement infection studies with standard (st) DENV preparations opsonized with anti-E mAbs in the presence or absence of furin inhibitor revealed that prM-containing particles present within st virus preparations contribute to antibody-dependent enhancement of infection. Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles.

Background

The inability of seasonal influenza vaccines to effectively protect against infection with antigenically drifted viruses or newly emerging pandemic viruses underlines the need for development of cross-reactive influenza vaccines that induce immunity against a variety of virus subtypes. Therefore, potential cross-protective vaccines, e.g., whole inactivated virus (WIV) vaccine, that can target conserved internal antigens such as the nucleoprotein (NP) and/or matrix protein (M1) need to be explored.

Methodology/Principal Findings

In the current study we show that a WIV vaccine, through induction of cross-protective cytotoxic T lymphocytes (CTLs), protects mice from heterosubtypic infection. This protection was abrogated after depletion of CD8+ cells in vaccinated mice, indicating that CTLs were the primary mediators of protection. Previously, we have shown that different procedures used for virus inactivation influence optimal activation of CTLs by WIV, most likely by affecting the membrane fusion properties of the virus. Specifically, inactivation with formalin (FA) severely compromises fusion activity of the virus, while inactivation with β-propiolactone (BPL) preserves fusion activity. Here, we demonstrate that vaccination of mice with BPL-inactivated H5N1 WIV vaccine induces solid protection from lethal heterosubtypic H1N1 challenge. By contrast, vaccination with FA-inactivated WIV, while preventing death after lethal challenge, failed to protect against development of disease and severe body weight loss. Vaccination with BPL-inactivated WIV, compared to FA-inactivated WIV, induced higher levels of specific CD8+ T cells in blood, spleen and lungs, and a higher production of granzyme B in the lungs upon H1N1 virus challenge.

Conclusion/Significance

The results underline the potential use of WIV as a cross-protective influenza vaccine candidate. However, careful choice of the virus inactivation procedure is important to retain membrane fusion activity and full immunogenicity of the vaccine.

Background

Each year rotavirus gastroenteritis results in thousands of paediatric hospitalisations and primary care visits in the Netherlands. While two vaccines against rotavirus are registered, routine immunisation of infants has not yet been implemented. Existing cost-effectiveness studies showed inconsistent results for these vaccines because of lack of consensus on the impact. We aimed to investigate which factors had a major impact on cost-effectiveness and were primarily responsible for the large differences in previously estimated cost-effectiveness ratios.

Methods

Based on updated data on health outcomes and cost estimates, we re-assessed the cost-effectiveness of routine paediatric rotavirus vaccination within the National Immunization Program for the Netherlands. Two consensus meetings were organised with national and international experts in the field to achieve consensus and resolve potential controversies.

Results

It was estimated that rotavirus vaccination in the Netherlands could avert 34,214 cases of rotavirus gastroenteritis in children aged less than 5 years. Notably, 2,779 hospitalisations were averted of which 315 were extensions of existing hospital stays due to nosocomial rotavirus infection. With a threshold varying from 20K€ - 50K€ per QALY and according to the base-case scenario, the full vaccination costs per child leading to cost-effectiveness was €57.76 -€77.71. Results were sensitive to the inclusion of potential vaccine induced herd protection, QALY losses and number of deaths associated with rotavirus gastroenteritis.

Conclusions

Our economic analysis indicates that inclusion of rotavirus vaccination in the Dutch National Immunization Program might be cost-effective depending on the cost of the vaccine and the impact of rotavirus gastroenteritis on children's quality of life.

Flaviviruses, such as dengue virus and West Nile virus, are enveloped viruses that infect cells through receptor-mediated endocytosis and fusion from within acidic endosomes. The cell entry process of flaviviruses is mediated by the viral E glycoprotein. This short review will address recent advances in the understanding of flavivirus cell entry with specific emphasis on the recent study of Zaitseva and coworkers, indicating that anionic lipids might play a crucial role in the fusion process of dengue virus [1].

Stockpiling of pre-pandemic influenza vaccines guarantees immediate vaccine availability to counteract an emerging pandemic. Generally, influenza vaccines need to be stored and handled refrigerated to prevent thermal degradation of the antigenic component. Requirement of a cold-chain, however, complicates stockpiling and the logistics of vaccine distribution. We, therefore, investigated the effect of elevated storage temperatures on the immunogenicity of a pre-pandemic influenza A H5N1 whole inactivated virus vaccine. Either suspended in liquid or kept as a freeze-dried powder, vaccines could be stored for 1 year at ambient temperature (20°C) with minimal loss of immunogenicity in mice. Elevation of the storage temperature to 40°C, however, resulted in a significant loss of immunogenic potency within 3 months if vaccines were stored in liquid suspension. In sharp contrast, freeze-dried powder formulations were stable at 40°C for at least 3 months. The presence of inulin or trehalose sugar excipients during freeze-drying of the vaccine proved to be critical to maintain its immunogenic potency during storage, and to preserve the characteristic Th1-type response to whole inactivated virus vaccine. These results indicate that whole inactivated virus vaccines may be stored and handled at room temperature in moderate climate zones for over a year with minimal decline and, if converted to dry-powder, even in hot climate zones for at least 3 months. The increased stability of dry-powder vaccine at 40°C may also point to an extended shelf-life when stored at 4°C. Use of the more stable dry-powder formulation could simplify stockpiling and thereby facilitating successful pandemic intervention.

Stockpiling of pre-pandemic influenza vaccines guarantees immediate vaccine availability to counteract an emerging pandemic. Generally, influenza vaccines need to be stored and handled refrigerated to prevent thermal degradation of the antigenic component. Requirement of a cold-chain, however, complicates stockpiling and the logistics of vaccine distribution. We, therefore, investigated the effect of elevated storage temperatures on the immunogenicity of a pre-pandemic influenza A H5N1 whole inactivated virus vaccine. Either suspended in liquid or kept as a freeze-dried powder, vaccines could be stored for 1 year at ambient temperature (20°C) with minimal loss of immunogenicity in mice. Elevation of the storage temperature to 40°C, however, resulted in a significant loss of immunogenic potency within 3 months if vaccines were stored in liquid suspension. In sharp contrast, freeze-dried powder formulations were stable at 40°C for at least 3 months. The presence of inulin or trehalose sugar excipients during freeze-drying of the vaccine proved to be critical to maintain its immunogenic potency during storage, and to preserve the characteristic Th1-type response to whole inactivated virus vaccine. These results indicate that whole inactivated virus vaccines may be stored and handled at room temperature in moderate climate zones for over a year with minimal decline and, if converted to dry-powder, even in hot climate zones for at least 3 months. The increased stability of dry-powder vaccine at 40°C may also point to an extended shelf-life when stored at 4°C. Use of the more stable dry-powder formulation could simplify stockpiling and thereby facilitating successful pandemic intervention.

Cells infected with dengue virus release a high proportion of immature prM-containing virions. In accordance, substantial levels of prM antibodies are found in sera of infected humans. Furthermore, it has been recently described that the rates of prM antibody responses are significantly higher in patients with secondary infection compared to those with primary infection. This suggests that immature dengue virus may play a role in disease pathogenesis. Interestingly, however, numerous functional studies have revealed that immature particles lack the ability to infect cells. In this report, we show that fully immature dengue particles become highly infectious upon interaction with prM antibodies. We demonstrate that prM antibodies facilitate efficient binding and cell entry of immature particles into Fc-receptor-expressing cells. In addition, enzymatic activity of furin is critical to render the internalized immature virus infectious. Together, these data suggest that during a secondary infection or primary infection of infants born to dengue-immune mothers, immature particles have the potential to be highly infectious and hence may contribute to the development of severe disease.

Author Summary

Dengue virus represents a major emerging arboviral pathogen circulating in the (sub)tropical regions of the world, putting 2.5 billion people at risk of infection. Each of the four circulating serotypes can cause disease ranging from febrile illness to devastating manifestations including dengue hemorrhagic fever and dengue shock syndrome. Severe illness is observed in individuals experiencing a re-infection with a heterologous dengue virus serotype and in infants born to dengue-immune mothers, presumably due to antibody-dependent enhancement of infection. Interestingly, it has been recently reported that patients experiencing a secondary infection have elevated levels of antibodies directed against the prM protein of immature dengue virus particles. Although it is known that cells infected with dengue virus release substantial amounts of prM-containing virions, numerous functional studies have demonstrated that immature particles lack the ability to infect cells. Herein, we show that essentially non-infectious fully immature dengue virions become virtually as infectious as wild type virus particles in the presence of prM antibodies. Anti-prM antibodies facilitate efficient binding and entry of immature dengue virus into cells carrying Fc-receptors. Furthermore, furin activity in target cells is critical for triggering infectivity of immature virus. These data indicate that immature dengue virus has the potential to be highly infectious and hence may contribute to disease pathogenesis.

West Nile virus (WNV) is a neurotropic flavivirus that is now a primary cause of epidemic encephalitis in North America. Studies of mice have demonstrated that the humoral immune response against WNV limits primary infection and protects against a secondary challenge. The most-potent neutralizing mouse monoclonal antibodies (MAbs) recognize an epitope on the lateral ridge of domain III (DIII-lr) of the envelope (E) protein. However, studies with serum from human patients show that antibodies against the DIII-lr epitope comprise, at best, a minor component of the human anti-WNV antibody response. Herein, we characterize in detail two WNV-specific human MAbs, CR4348 and CR4354, that were isolated from B-cell populations of convalescent patients. These MAbs strongly neutralize WNV infection of cultured cells, protect mice against lethal infection in vivo, and yet poorly recognize recombinant forms of the E protein. Instead, CR4348 and CR4354 bind determinants on intact WNV virions and subviral particles in a pH-sensitive manner, and neutralization is altered by mutations at the dimer interface in domain II and the hinge between domains I and II, respectively. CR4348 and CR4354 human MAbs neutralize infection at a postattachment step in the viral life cycle, likely by inhibiting acid-induced fusion within the endosome.

Defining the precise cellular mechanisms of neutralization by potently inhibitory antibodies is important for understanding how the immune system successfully limits viral infections. We recently described a potently inhibitory monoclonal antibody (MAb E16) against the envelope (E) protein of West Nile virus (WNV) that neutralizes infection even after virus has spread to the central nervous system. Herein, we define its mechanism of inhibition. E16 blocks infection primarily at a post-attachment step as antibody-opsonized WNV enters permissive cells but cannot escape from endocytic compartments. These cellular experiments suggest that E16 blocks the acid-catalyzed fusion step that is required for nucleocapsid entry into the cytoplasm. Indeed, E16 directly inhibits fusion of WNV with liposomes. Additionally, low-pH exposure of E16–WNV complexes in the absence of target membranes did not fully inactivate infectious virus, further suggesting that E16 prevents a structural transition required for fusion. Thus, a strongly neutralizing anti–WNV MAb with therapeutic potential is potently inhibitory because it blocks viral fusion and thereby promotes clearance by delivering virus to the lysosome for destruction.

Author Summary

Antibodies are essential components of the immune response against many pathogens, including viruses. A greater understanding of the mechanisms by which the most strongly inhibitory antibodies act may influence the design and production of novel vaccines or antibody-based therapies. Our group recently generated a highly inhibitory monoclonal antibody (E16) against the envelope protein of West Nile virus, which can abort infection in animals even after the virus has spread to the brain. In this paper, we define its mechanism of action. We show that E16 blocks infection by preventing West Nile virus from transiting from endosomes, an obligate step in the entry pathway of the viral lifecycle. Thus, a strongly inhibitory anti–West Nile virus antibody is highly neutralizing because it blocks fusion and delivers virus to the lysosome for destruction.

Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments.

Author Summary

Dengue virus (DENV) is the most common arthropod-borne infection worldwide with 50–100 million cases annually. Despite its high clinical impact, little is known about the infectious cell entry pathway of the virus. Previous studies have shown conflicting evidence about whether the virus fuses directly with the cell plasma membrane or enters cells by receptor-mediated endocytosis. In this manuscript, we dissect the cell entry pathway of DENV by tracking single fluorescently labeled DENV particles in living cells expressing various fluorescent cellular markers, using real-time multi-color fluorescence microscopy. We show that DENV particles are delivered to pre-existing clathrin-coated pits by diffusion along the cell surface. Following clathrin-mediated uptake, the majority of DENV particles are transported to early endosomes, which mature into late endosomes, where membrane fusion occurs. This is the first study that describes the cell entry process of DENV at the single particle level and therefore provides unique mechanistic and kinetic insights into the route of entry, endocytic trafficking behavior, and membrane fusion properties of individual DENV particles in living cells. This paper opens new avenues in flavivirus biology and will lead toward a better understanding of the critical determinants in DENV infection.

In the case of an influenza pandemic, the current global influenza vaccine production capacity will be unable to meet the demand for billions of vaccine doses. The ongoing threat of an H5N1 pandemic therefore urges the development of highly immunogenic, dose-sparing vaccine formulations. In unprimed individuals, inactivated whole virus (WIV) vaccines are more immunogenic and induce protective antibody responses at a lower antigen dose than other formulations like split virus (SV) or subunit (SU) vaccines. The reason for this discrepancy in immunogenicity is a long-standing enigma. Here, we show that stimulation of Toll-like receptors (TLRs) of the innate immune system, in particular stimulation of TLR7, by H5N1 WIV vaccine is the prime determinant of the greater magnitude and Th1 polarization of the WIV-induced immune response, as compared to SV- or SU-induced responses. This TLR dependency largely explains the relative loss of immunogenicity in SV and SU vaccines. The natural pathogen-associated molecular pattern (PAMP) recognized by TLR7 is viral genomic ssRNA. Processing of whole virus particles into SV or SU vaccines destroys the integrity of the viral particle and leaves the viral RNA prone to degradation or involves its active removal. Our results show for a classic vaccine that the acquired immune response evoked by vaccination can be enhanced and steered by the innate immune system, which is triggered by interaction of an intrinsic vaccine component with a pattern recognition receptor (PRR). The insights presented here may be used to further improve the immune-stimulatory and dose-sparing properties of classic influenza vaccine formulations such as WIV, and will facilitate the development of new, even more powerful vaccines to face the next influenza pandemic.

Author Summary

The rise and spread of the highly pathogenic avian H5N1 influenza virus has seriously increased the risk of a new influenza pandemic. However, the number of vaccine doses that can be produced with today's production capacity will fall short of the demand in times of a pandemic. Use of inactivated whole virus (WIV) vaccines, which are more immunogenic than split virus or subunit vaccines in an unprimed population, could contribute to a dose-sparing strategy. Yet, the mechanisms underlying the superior immunogenicity of WIV vaccine formulations are unknown. Here, we demonstrate that the viral RNA present in inactivated virus particles is crucial for the improved immunogenic properties of WIV in mice. By triggering Toll-like receptor 7 (TLR7), the viral RNA activates innate immune mechanisms that augment and determine subsequent adaptive responses. Efficient TLR7 signalling is lost in split virus and subunit vaccines with the processing steps that lead to disruption of the integrity of the virus particle and exclusion of the RNA. Our results prove for the first time to our knowledge that the immune-potentiating mechanism of a classic vaccine is based on activation of the innate immune system by one of its structural components. These findings may reflect a general principle for viral vaccines and provide a rational basis for further improvement of influenza vaccines, which are urgently needed in the face of the current H5N1 pandemic threat.

In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain S1 on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by infectivity assays, leading to an infectious unit-to-particle ratio of approximately 1:2,600 to 1:72,000, depending on the specific assays used. In order to explain this high ratio, we investigated the receptor binding and membrane fusion characteristics of single DENV particles in living cells using real-time fluorescence microscopy. For this purpose, DENV was labeled with the lipophilic fluorescent probe DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt). The surface density of the DiD dye in the viral membrane was sufficiently high to largely quench the fluorescence intensity but still allowed clear detection of single virus particles. Fusion of the viral membrane with the cell membrane was evident as fluorescence dequenching. It was observed that DENV binds very inefficiently to the cells used, explaining at least in part the high infectious unit-to-particle ratio. The particles that did bind to the cells showed different types of transport behavior leading to membrane fusion in both the periphery and perinuclear regions of the cell. Membrane fusion was observed in 1 out of 6 bound virus particles, indicating that a substantial fraction of the virus has the capacity to fuse. DiD dequenching was completely inhibited by ammonium chloride, demonstrating that fusion occurs exclusively from within acidic endosomes.

Previously, it has been shown that the exposure of Semliki Forest virus (SFV) to a mildly acidic environment induces a rapid and complete loss of the ability of the virus to bind and fuse to target membranes added subsequently. In the present study, incubation of SFV at low pH followed by a specific reneutralization step resulted in a partial reversion of this loss of viral fusion capacity, as assessed in a liposomal model system. Also, the ability of the viral E1 fusion protein to undergo liposome-stimulated trimerization was restored. Furthermore, acid-treated and neutralized SFV largely retained infectivity. Exposure of SFV to low pH induced dissociation of the E1/E2 heterodimer, which was not reversed upon neutralization. It is concluded that the SFV E1 fusion protein, after acid-induced dissociation from E2, rapidly adopts an intermediate, nontrimeric conformation in which it is no longer able to interact with target membrane lipids. Neutralization restores the ability of E1 to interact with membranes. This interaction, however, remains strictly dependent on low pH.

Passage of Sindbis virus (SIN) in BHK-21 cells has been shown to select for virus mutants with high affinity for the glycosaminoglycan heparan sulfate (HS). Three loci in the viral spike protein E2 (E2:1, E2:70, and E2:114) have been identified that mutate during adaptation and independently confer on the virus the ability to bind to cell surface HS (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). In this study, we used HS-adapted SIN mutants to evaluate a new model system involving target liposomes containing lipid-conjugated heparin (HepPE) as an HS receptor analog for the virus. HS-adapted SIN, but not nonadapted wild-type SIN TR339, interacted efficiently with HepPE-containing liposomes at neutral pH. Binding was competitively inhibited by soluble heparin. Despite the efficient binding of HS-adapted SIN to HepPE-containing liposomes at neutral pH, there was no fusion under these conditions. Fusion did occur, however, at low pH, consistent with cellular entry of the virus via acidic endosomes. At low pH, wild-type or HS-adapted SIN underwent fusion with liposomes with or without HepPE with similar kinetics, suggesting that interaction with the HS receptor analog at neutral pH has little influence on subsequent fusion of SIN at low pH. Finally, Semliki Forest virus (SFV), passaged frequently on BHK-21 cells, also interacted efficiently with HepPE-containing liposomes, indicating that SFV, like other alphaviruses, readily adapts to cell surface HS. In conclusion, the liposomal model system presented in this paper may serve as a novel tool for the study of receptor interactions and membrane fusion properties of HS-interacting enveloped viruses.

The amino acid at position 55 of the E2 glycoprotein (E255) of Sindbis virus (SV) is a critical determinant of SV neurovirulence in mice. Recombinant virus strain TE (E255 = histidine) differs only at this position from virus strain 633 (E255= glutamine), yet TE is considerably more neurovirulent than 633. TE replicates better than 633 in a neuroblastoma cell line (N18), but similarly in BHK cells. Immunofluorescence staining showed that most N18 cells were infected by TE at a multiplicity of infection (MOI) of 50 to 500 and by 633 only at an MOI of 5,000, while both viruses infected essentially 100% of BHK cells at an MOI of 5. When exposed to pH 5, TE and 633 viruses fused to similar extents with liposomes derived from BHK or N18 cell lipids, but fusion with N18-derived liposomes was less extensive (15 to 20%) than fusion with BHK-derived liposomes (∼50%). Binding of TE and 633 to N18, but not BHK, cells was dependent on the medium used for virus binding. Differences between TE and 633 binding to N18 cells were evident in Dulbecco's modified Eagle medium (DMEM), but not in RPMI. In DMEM, the binding efficiency of 633 decreased significantly as the pH was raised from 6.5 to 8.0, while that of TE did not change. The same pattern was observed with RPMI when the ionic strength of RPMI was increased to that of DMEM. TE bound better to heparin-Sepharose than 633, but this difference was not pH dependent. Growth of N18 and BHK cells in sodium chlorate to eliminate all sulfation decreased virus-cell binding, suggesting the involvement of sulfated molecules on the cell surface. Taken together, the presence of glutamine at E255 impairs SV binding to neural cells under conditions characteristic of interstitial fluid. We conclude that mutation to histidine participates in or stabilizes the interaction between the virus and the surface of neural cells, contributing to greater neurovirulence.

The spike glycoprotein E2 of Sindbis virus (SIN) is synthesized in the infected cell as a PE2 precursor protein, which matures through cleavage by a cellular furin-like protease. Previous work has shown that SIN mutants impaired in PE2 cleavage are noninfectious on BHK-21 cells, the block in infection being localized at a step after virus-receptor interaction but prior to RNA replication. Here, we studied the membrane fusion properties of SIN PE2 cleavage mutants and observed that these viruses are impaired in their ability to form an E1 homotrimer and to fuse with liposomes at a mildly acidic pH. The block in spike rearrangement and fusion could be overridden by exposure of the mutant viruses to very low pH (<4.5). Cleavage mutants with second-site resuscitating mutations in PE2 were highly infectious for BHK-21 cells. The ability of these viruses to form E1 homotrimers and to fuse at a mildly acidic pH was completely restored despite a sustained lack of PE2 cleavage.

Membrane fusion mediated by influenza virus hemagglutinin (HA) is believed to proceed via the cooperative action of multiple HA trimers. To determine the minimal number of HA trimers required to trigger fusion, and to assess the importance of cooperativity between these HA trimers, we have generated virosomes containing coreconstituted HAs derived from two strains of virus with different pH dependencies for fusion, X-47 (optimal fusion at pH 5.1; threshold at pH 5.6) and A/Shangdong (optimal fusion at pH 5.6; threshold at pH 6.0), and measured fusion of these virosomes with erythrocyte ghosts by a fluorescence lipid mixing assay. Virosomes with different X-47-to-A/Shangdong HA ratios, at a constant HA-to-lipid ratio, showed comparable ghost-binding activities, and the low-pH-induced conformational change of A/Shangdong HA did not affect the fusion activity of X-47 HA. The initial rate of fusion of these virosomes at pH 5.7 increased directly proportional to the surface density of A/Shangdong HA, and a single A/Shangdong trimer per virosome appeared to suffice to induce fusion. The reciprocal of the lag time before the onset of fusion was directly proportional to the surface density of fusion-competent HA. These results support the notion that there is no cooperativity between HA trimers during influenza virus fusion.

There is controversy as to whether the cell entry mechanism of Sindbis virus (SIN) involves direct fusion of the viral envelope with the plasma membrane at neutral pH or uptake by receptor-mediated endocytosis and subsequent low-pH-induced fusion from within acidic endosomes. Here, we studied the membrane fusion activity of SIN in a liposomal model system. Fusion was followed fluorometrically by monitoring the dilution of pyrene-labeled lipids from biosynthetically labeled virus into unlabeled liposomes or from labeled liposomes into unlabeled virus. Fusion was also assessed on the basis of degradation of the viral core protein by trypsin encapsulated in the liposomes. SIN fused efficiently with receptor-free liposomes, consisting of phospholipids and cholesterol, indicating that receptor interaction is not a mechanistic requirement for fusion of the virus. Fusion was optimal at pH 5.0, with a threshold at pH 6.0, and undetectable at neutral pH, supporting a cell entry mechanism of SIN involving fusion from within acidic endosomes. Under optimal conditions, 60 to 85% of the virus fused, depending on the assay used, corresponding to all of the virus bound to the liposomes as assessed in a direct binding assay. Preincubation of the virus alone at pH 5.0 resulted in a rapid loss of fusion capacity. Fusion of SIN required the presence of both cholesterol and sphingolipid in the target liposomes, cholesterol being primarily involved in low-pH-induced virus-liposome binding and the sphingolipid catalyzing the fusion process itself. Under low-pH conditions, the E2/E1 heterodimeric envelope glycoprotein of the virus dissociated, with formation of a trypsin-resistant E1 homotrimer, which kinetically preceded the fusion reaction, thus suggesting that the E1 trimer represents the fusion-active conformation of the viral spike.