One More Piece in the VACV Ecological Puzzle: Could Peridomestic Rodents Be the Link between Wildlife and Bovine Vaccinia Outbreaks in Brazil?

Background

Despite the fact that smallpox eradication was declared by the World Health Organization (WHO) in 1980, other poxviruses have emerged and re-emerged, with significant public health and economic impacts. Vaccinia virus (VACV), a poxvirus used during the WHO smallpox vaccination campaign, has been involved in zoonotic infections in Brazilian rural areas (Bovine Vaccinia outbreaks – BV), affecting dairy cattle and milkers. Little is known about VACV''s natural hosts and its epidemiological and ecological characteristics. Although VACV was isolated and/or serologically detected in Brazilian wild animals, the link between wildlife and farms has not yet been elucidated.

Methodology/Principal Findings

In this study, we describe for the first time, to our knowledge, the isolation of a VACV (Mariana virus - MARV) from a mouse during a BV outbreak. Genetic data, in association with biological assays, showed that this isolate was the same etiological agent causing exanthematic lesions observed in the cattle and human inhabitants of a particular BV-affected area. Phylogenetic analysis grouped MARV with other VACV isolated during BV outbreaks.

Conclusion/Significance

These data provide new biological and epidemiological information on VACV and lead to an interesting question: could peridomestic rodents be the link between wildlife and BV outbreaks?     

Preexisting Japanese encephalitis virus neutralizing antibodies and increased symptomatic dengue illness in a school-based cohort in Thailand

Background

Dengue viruses (DENVs) and Japanese encephalitis virus (JEV) have significant cross-reactivity in serological assays; the clinical implications of this remain undefined. An improved understanding of whether and how JEV immunity modulates the clinical outcome of DENV infection is important as large-scale DENV vaccine trials will commence in areas where JEV is co-endemic and/or JEV immunization is routine.

Methods and Findings

The association between preexisting JEV neutralizing antibodies (NAbs) and the clinical severity of DENV infection was evaluated in a prospective school-based cohort in Thailand that captured asymptomatic, non-hospitalized, and hospitalized DENV infections. Covariates considered included age, baseline DENV antibody status, school of attendance, epidemic year, and infecting DENV serotype. 942 children experienced at least one DENV infection between 1998 and 2002, out of 3,687 children who were enrolled for at least one full year. In crude analysis, the presence of JEV NAbs was associated with an increased occurrence of symptomatic versus asymptomatic infection (odds ratio [OR] = 1.55, 95% CI: 1.08–2.23) but not hospitalized illness or dengue hemorrhagic fever (DHF). The association was strongest in children with negative DENV serology (DENV-naive) (OR = 2.75, 95% CI: 1.12–6.72), for whom the presence of JEV NAbs was also associated with a symptomatic illness of longer duration (5.4 days for JEV NAb+ versus 2.6 days for JEV NAb-, p = 0.048). JEV NAbs were associated with increased DHF in younger children with multitypic DENV NAb profiles (OR = 4.05, 95% CI: 1.18 to 13.87). Among those with JEV NAbs, the association with symptomatic illness did not vary by antibody titer.

Interpretation

The prior existence of JEV NAbs was associated with an increased probability of symptomatic as compared to asymptomatic DENV illness. These findings are in contrast to previous studies suggesting an attenuating effect of heterologous flavivirus immunity on DENV disease severity.     

A recombinant vaccine effectively induces c5a-specific neutralizing antibodies and prevents arthritis

Objectives

To develop and validate a recombinant vaccine to attenuate inflammation in arthritis by sustained neutralization of the anaphylatoxin C5a.

Methods

We constructed and expressed fusion protein of C5a and maltose binding protein. Efficacy of specific C5a neutralization was tested using the fusion protein as vaccine in three different arthritis mouse models: collagen induced arthritis (CIA), chronic relapsing CIA and collagen antibody induced arthritis (CAIA). Levels of anti-C5a antibodies and anti-collagen type II were measured by ELISA. C5a neutralization assay was done using a rat basophilic leukemia cell-line transfected with the human C5aR. Complement activity was determined using a hemolytic assay and joint morphology was assessed by histology.

Results

Vaccination of mice with MBP-C5a led to significant reduction of arthritis incidence and severity but not anti-collagen antibody synthesis. Histology of the MBP-C5a and control (MBP or PBS) vaccinated mice paws confirmed the vaccination effect. Sera from the vaccinated mice developed C5a-specific neutralizing antibodies, however C5 activation and formation of the membrane attack complex by C5b were not significantly altered.

Conclusions

Exploitation of host immune response to generate sustained C5a neutralizing antibodies without significantly compromising C5/C5b activity is a useful strategy for developing an effective vaccine for antibody mediated and C5a dependent inflammatory diseases. Further developing of such a therapeutic vaccine would be more optimal and cost effective to attenuate inflammation without affecting host immunity.     

Vaccinia Virus Induces Rapid Necrosis in Keratinocytes by a STAT3-Dependent Mechanism

Rationale

Humans with a dominant negative mutation in STAT3 are susceptible to severe skin infections, suggesting an essential role for STAT3 signaling in defense against cutaneous pathogens.

Methods

To focus on innate antiviral defenses in keratinocytes, we used a standard model of cutaneous infection of severe combined immunodeficient mice with the current smallpox vaccine, ACAM-2000. In parallel, early events post-infection with the smallpox vaccine ACAM-2000 were investigated in cultured keratinocytes of human and mouse origin.

Results

Mice treated topically with a STAT3 inhibitor (Stattic) developed larger vaccinia lesions with higher virus titers and died more rapidly than untreated controls. Cultured human and murine keratinocytes infected with ACAM-2000 underwent rapid necrosis, but when treated with Stattic or with inhibitors of RIP1 kinase or caspase-1, they survived longer, produced higher titers of virus, and showed reduced activation of type I interferon responses and inflammatory cytokines release. Treatment with inhibitors of RIP1 kinase and STAT3, but not caspase-1, also reduced the inflammatory response of keratinocytes to TLR ligands. Vaccinia growth properties in Vero cells, which are known to be defective in some antiviral responses, were unaffected by inhibition of RIP1K, caspase-1, or STAT3.

Conclusions

Our findings indicate that keratinocytes suppress the replication and spread of vaccinia virus by undergoing rapid programmed cell death, in a process requiring STAT3. These data offer a new framework for understanding susceptibility to skin infection in patients with STAT3 mutations. Interventions which promote prompt necroptosis/pyroptosis of infected keratinocytes may reduce risks associated with vaccination with live vaccinia virus.     

Envelope variable region 4 is the first target of neutralizing antibodies in early simian immunodeficiency virus mac251 infection of rhesus monkeys

A major goal of AIDS vaccine development is to design vaccination strategies that can elicit broad and potent protective antibodies. The initial viral targets of neutralizing antibodies (NAbs) early after human or simian immunodeficiency virus (HIV/SIV) infection are not known. The identification of early NAb epitopes that induce protective immunity or retard the progression of disease is important for AIDS vaccine development. The aim of this study was to determine the Env residues targeted by early SIV NAbs and to assess the influence of prior vaccination on neutralizing antibody kinetics and specificity during early infection. We previously described stereotypic env sequence variations in SIVmac251-infected rhesus monkeys that resulted in viral escape from NAbs. Here, we defined the early viral targets of neutralization and determined whether the ability of serum antibody from infected monkeys to neutralize SIV was altered in the setting of prior vaccination. To localize the viral determinants recognized by early NAbs, a panel of mutant pseudoviruses was assessed in a TZM-bl reporter gene neutralization assay to define the precise changes that eliminate recognition by SIV Env-specific NAbs in 16 rhesus monkeys. Changing R420 to G or R424 to Q in V4 of Env resulted in the loss of recognition by NAbs in vaccinated monkeys. In contrast, mutations in the V1 region of Env did not alter the NAb profile. These findings indicate that early NAbs are directed toward SIVmac251 Env V4 but not the V1 region, and that this env vaccination regimen did not alter the kinetics or the breadth of NAbs during early infection.     

Human immunodeficiency virus type 1 superinfection occurs despite relatively robust neutralizing antibody responses

Superinfection by a second human immunodeficiency virus type 1 (HIV-1) strain indicates that gaps in protective immunity occur during natural infection. To define the role of HIV-1-specific neutralizing antibodies (NAbs) in this setting, we examined NAb responses in 6 women who became superinfected between ~1 to 5 years following initial infection compared to 18 women with similar risk factors who did not. Although superinfected individuals had less NAb breadth than matched controls at ~1 year postinfection, no significant differences in the breadth or potency of NAb responses were observed just prior to the second infection. In fact, four of the six subjects had relatively broad and potent NAb responses prior to infection by the second strain. To more specifically examine the specificity of the NAbs against the superinfecting virus, these variants were cloned from five of the six individuals. The superinfecting variants did not appear to be inherently neutralization resistant, as measured against a pool of plasma from unrelated HIV-infected individuals. Moreover, the superinfected individuals were able to mount autologous NAb responses to these variants following reinfection. In addition, most superinfected individuals had NAbs that could neutralize their second viral strains prior to their reinfection, suggesting that the level of NAbs elicited during natural infection was not sufficient to block infection. These data indicate that preventing infection by vaccination will likely require broader and more potent NAb responses than those found in HIV-1-infected individuals.     

Protection in macaques immunized with HIV-1 candidate vaccines can be predicted using the kinetics of their neutralizing antibodies

Background

A vaccine is needed to control the spread of human immunodeficiency virus type 1 (HIV-1). An in vitro assay that can predict the protection induced by a vaccine would facilitate the development of such a vaccine. A potential candidate would be an assay to quantify neutralization of HIV-1.

Methods and Findings

We have used sera from rhesus macaques that have been immunized with HIV candidate vaccines and subsequently challenged with simian human immunodeficiency virus (SHIV). We compared neutralization assays with different formats. In experiments with the standardized and validated TZMbl assay, neutralizing antibody titers against homologous SHIV_SF162P4 pseudovirus gave a variable correlation with reductions in plasma viremia levels. The target cells used in the assays are not just passive indicators of virus infection but are actively involved in the neutralization process. When replicating virus was used with GHOST cell assays, events during the absorption phase, as well as the incubation phase, determine the level of neutralization. Sera that are associated with protection have properties that are closest to the traditional concept of neutralization: the concentration of antibody present during the absorption phase has no effect on the inactivation rate. In GHOST assays, events during the absorption phase may inactivate a fixed number, rather than a proportion, of virus so that while complete neutralization can be obtained, it can only be found at low doses particularly with isolates that are relatively resistant to neutralization.

Conclusions

Two scenarios have the potential to predict protection by neutralizing antibodies at concentrations that can be induced by vaccination: antibodies that have properties close to the traditional concept of neutralization may protect against a range of challenge doses of neutralization sensitive HIV isolates; a window of opportunity also exists for protection against isolates that are more resistant to neutralization but only at low challenge doses.     

Immunomodulator-Based Enhancement of Anti Smallpox Immune Responses

Background

The current live vaccinia virus vaccine used in the prevention of smallpox is contraindicated for millions of immune-compromised individuals. Although vaccination with the current smallpox vaccine produces protective immunity, it might result in mild to serious health complications for some vaccinees. Thus, there is a critical need for the production of a safe virus-free vaccine against smallpox that is available to everyone. For that reason, we investigated the impact of imiquimod and resiquimod (Toll-like receptors agonists), and the codon-usage optimization of the vaccinia virus A27L gene in the enhancement of the immune response, with intent of producing a safe, virus-free DNA vaccine coding for the A27 vaccinia virus protein.

Methods

We analyzed the cellular-immune response by measuring the IFN-γ production of splenocytes by ELISPOT, the humoral-immune responses measuring total IgG and IgG2a/IgG1 ratios by ELISA, and the TH1 and TH2 cytokine profiles by ELISA, in mice immunized with our vaccine formulation.

Results

The proposed vaccine formulation enhanced the A27L vaccine-mediated production of IFN-γ on mouse spleens, and increased the humoral immunity with a TH1-biased response. Also, our vaccine induced a TH1 cytokine milieu, which is important against viral infections.

Conclusion

These results support the efforts to find a new mechanism to enhance an immune response against smallpox, through the implementation of a safe, virus-free DNA vaccination platform.     

Original Encounter with Antigen Determines Antigen-Presenting Cell Imprinting of the Quality of the Immune Response in Mice

Background

Obtaining a certain multi-functionality of cellular immunity for the control of infectious diseases is a burning question in immunology and in vaccine design. Early events, including antigen shuttling to secondary lymphoid organs and recruitment of innate immune cells for adaptive immune response, determine host responsiveness to antigens. However, the sequence of these events and their impact on the quality of the immune response remain to be elucidated. Here, we chose to study Modified Vaccinia virus Ankara (MVA) which is now replacing live Smallpox vaccines and is proposed as an attenuated vector for vaccination strategies against infectious diseases.

Methodology/Principal findings

We analyzed in vivo mechanisms triggered following intradermal (i.d.) and intramuscular (i.m.) Modified Vaccinia virus Ankara (MVA) administration. We demonstrated significant differences in the antigen shuttling to lymphoid organs by macrophages (MΦs), myeloid dendritic cells (DCs), and neutrophils (PMNs). MVA i.d. administration resulted in better antigen distribution and more sustained antigen-presenting cells (APCs) recruitment into draining lymph nodes than with i.m. administration. These APCs, which comprise both DCs and MΦs, were differentially involved in T cell priming and shaped remarkably the quality of cytokine-producing virus-specific T cells according to the entry route of MVA.

Conclusions/Significance

This study improves our understanding of the mechanisms of antigen delivery and their consequences on the quality of immune responses and provides new insights for vaccine development.     

International Network for Comparison of HIV Neutralization Assays: The NeutNet Report

Background

Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1) vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration (NeutNet) involving 18 independent participants was organized to compare different assays.

Methods

Each laboratory evaluated four neutralizing reagents (TriMab, 447-52D, 4E10, sCD4) at a given range of concentrations against a panel of 11 viruses representing a wide range of genetic subtypes and phenotypes. A total of 16 different assays were compared. The assays utilized either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (virus infectivity assays, VI assays), or their Env-pseudotyped (gp160) derivatives produced in 293T cells (PSV assays) from molecular clones or uncloned virus. Target cells included PBMC and genetically-engineered cell lines in either a single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs that included extracellular or intracellular p24 antigen detection, RNA quantification and luciferase and beta-galactosidase reporter gene expression.

Findings

PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus and inhibitor used. For example, for TriMab, the mean IC50 was always lower in PSV than in VI assays. However, with 4E10 or sCD4 some viruses were neutralized with a lower IC50 in VI assays than in the PSV assays. Inter-laboratory concordance was slightly better for PSV than for VI assays with some viruses, but for other viruses agreement between laboratories was limited and depended on both the virus and the neutralizing reagent.

Conclusions

The NeutNet project demonstrated clear differences in assay sensitivity that were dependent on both the neutralizing reagent and the virus. No single assay was capable of detecting the entire spectrum of neutralizing activities. Since it is not known which in vitro assay correlates with in vivo protection, a range of neutralization assays is recommended for vaccine evaluation.     

Detection of Extensive Cross-Neutralization between Pandemic and Seasonal A/H1N1 Influenza Viruses Using a Pseudotype Neutralization Assay

Background

Cross-immunity between seasonal and pandemic A/H1N1 influenza viruses remains uncertain. In particular, the extent that previous infection or vaccination by seasonal A/H1N1 viruses can elicit protective immunity against pandemic A/H1N1 is unclear.

Methodology/Principal Findings

Neutralizing titers against seasonal A/H1N1 (A/Brisbane/59/2007) and against pandemic A/H1N1 (A/California/04/2009) were measured using an HIV-1-based pseudovirus neutralization assay. Using this highly sensitive assay, we found that a large fraction of subjects who had never been exposed to pandemic A/H1N1 express high levels of pandemic A/H1N1 neutralizing titers. A significant correlation was seen between neutralization of pandemic A/H1N1 and neutralization of a standard seasonal A/H1N1 strain. Significantly higher pandemic A/H1N1 neutralizing titers were measured in subjects who had received vaccination against seasonal influenza in 2008–2009. Higher pandemic neutralizing titers were also measured in subjects over 60 years of age.

Conclusions/Significance

Our findings reveal that the extent of protective cross-immunity between seasonal and pandemic A/H1N1 influenza viruses may be more important than previously estimated. This cross-immunity could provide a possible explanation of the relatively mild profile of the recent influenza pandemic.     

Antibody responses against xenotropic murine leukemia virus-related virus envelope in a murine model

Background

Xenotropic murine leukemia virus-related virus (XMRV) was recently discovered to be the first human gammaretrovirus that is associated with chronic fatigue syndrome and prostate cancer (PC). Although a mechanism for XMRV carcinogenesis is yet to be established, this virus belongs to the family of gammaretroviruses well known for their ability to induce cancer in the infected hosts. Since its original identification XMRV has been detected in several independent investigations; however, at this time significant controversy remains regarding reports of XMRV detection/prevalence in other cohorts and cell type/tissue distribution. The potential risk of human infection, coupled with the lack of knowledge about the basic biology of XMRV, warrants further research, including investigation of adaptive immune responses. To study immunogenicity in vivo, we vaccinated mice with a combination of recombinant vectors expressing codon-optimized sequences of XMRV gag and env genes and virus-like particles (VLP) that had the size and morphology of live infectious XMRV.

Results

Immunization elicited Env-specific binding and neutralizing antibodies (NAb) against XMRV in mice. The peak titers for ELISA-binding antibodies and NAb were 1∶1024 and 1∶464, respectively; however, high ELISA-binding and NAb titers were not sustained and persisted for less than three weeks after immunizations.

Conclusions

Vaccine-induced XMRV Env antibody titers were transiently high, but their duration was short. The relatively rapid diminution in antibody levels may in part explain the differing prevalences reported for XMRV in various prostate cancer and chronic fatigue syndrome cohorts. The low level of immunogenicity observed in the present study may be characteristic of a natural XMRV infection in humans.     

Heterosubtypic antibody response elicited with seasonal influenza vaccine correlates partial protection against highly pathogenic H5N1 virus

Background

The spread of highly pathogenic avian influenza (HPAI) H5N1 virus in human remains a global health concern. Heterosubtypic antibody response between seasonal influenza vaccine and potential pandemic influenza virus has important implications for public health. Previous studies by Corti et al. and by Gioia et al. demonstrate that heterosubtypic neutralizing antibodies against the highly pathogenic H5N1 virus can be elicited with a seasonal influenza vaccine in humans. However, whether such response offers immune protection against highly pathogenic H5N1 virus remained to be determined.

Methodology/Principal Findings

In this study, using a sensitive influenza HA (hemagglutinin) and NA (neuraminidase) pseudotype-based neutralization (PN) assay we first confirmed that low levels of heterosubtypic neutralizing antibody response against H5N1 virus were indeed elicited with seasonal influenza vaccine in humans. We then immunized mice with the seasonal influenza vaccine and challenged them with lethal doses of highly pathogenic H5N1 virus. As controls, we immunized mice with homosubtypic H5N1 virus like particles (VLP) or PBS and challenged them with the same H5N1 virus. Here we show that low levels of heterosubtypic neutralizing antibody response were elicited with seasonal influenza vaccine in mice, which were significantly higher than those in PBS control. Among them 2 out of 27 whose immune sera exhibited similar levels of neutralizing antibody response as VLP controls actually survived from highly pathogenic H5N1 virus challenge.

Conclusions/Significance

Therefore, we conclude that low levels of heterosubtypic neutralizing antibody response are indeed elicited with seasonal influenza vaccine in humans and mice and at certain levels such response offers immune protection against severity of H5N1 virus infection.     

Comparison on Virulence and Immunogenicity of Two Recombinant Vaccinia Vaccines,Tian Tan and Guang9 Strains,Expressing the HIV-1 Envelope Gene

Background

The vaccinia virus Guang9 strain (VG9), derived from the vaccinia virus Tian Tan strain (VTT) has been found to be less virulent than VTT.

Methodology/Principal Findings

To investigate whether VG9 could be a potential replicating virus vector, the TK genes in VG9 and VTT were replaced with the HIV-1 envelope gene via homologous recombination, resulting in the recombinant viruses, VG9-E and VTT-E. The biology, virulence, humoral and cellular immunological responses of VG9-E and VTT-E were evaluated. Our results indicated no obvious difference in range of host cells and diffusion between two recombinant viruses. Neurovirulence for VG9-E in weanling and suckling mice, and skin virulence in rabbits, were lower than that of VTT-E. The humoral immune responses, including binding antibody and neutralizing antibody responses, induced by VG9-E were not significantly different from those for VTT-E whilst IFN-γ response which represented cellular immune response induced by VG9-E was significantly higher than that did by VTT-E.

Conclusions/Significance

Our results indicated that VG9-E was less virulent, yet induced higher cellular immune response than VTT-E. Therefore, it could be an ideal replicating vaccinia vector for HIV vaccine research and development.     

Demographic and Clinical Factors Associated with Response to Smallpox Vaccine in Preimmunized Volunteers

Context

In March 2003, the French Ministry of Health implemented a program on preparedness and response to a biological attack using smallpox as weapon. This program included the establishment of a preoutbreak national team that could be revaccinated against smallpox.

Objective

To identify demographic and clinical factors associated with vaccination success defined as the presence of a pustule at the inoculation site at day 8 (days 7–9), with an undiluted vaccinia virus derived from a Lister strain among preimmunized volunteers.

Volunteers and Methods

From March 2003 to November 2006, we have studied prospectively 226 eligible volunteers. Demographic data were recorded for each volunteer (age, sex, number of previously smallpox vaccinations and date of the last vaccination). Smallpox vaccine adverse reactions were diagnosed on the basis of clinical examination performed at days 0, 7, 14, 21 and 28 after revaccination.

Results

A total of 226 volunteers (sex ratio H/F = 2.7) were revaccinated. Median age was 45 years (range: 27–63 yrs). All volunteers completed follow-up. Median number of vaccinations before revaccination was 2 (range: 1–8). The median delay between time of the study and the last vaccination was 29 years (range; 18–60 yrs). Sixty-one volunteers (27%) experienced one (n = 40) or more (n = 21) minor side effects during the 2–14 days after revaccination. Successful vaccination was noted in 216/226 volunteers (95.6%) at day 8 and the median of the pustule diameter was 5 mm (range: 1–20 mm). Size of the pustule at day 8 was correlated with age (p = 0.03) and with the presence of axillary adenopathy after revaccination (p = 0.007). Sex, number of prior vaccinations, delay between the last vaccination and revaccination, and local or systemic side effects with the exception of axillary adenopathy, were not correlated with the size of the pustule at day 8.

Conclusions

Previously vaccinated volunteers can be successfully revaccinated with the Lister strain.     

Tiered Categorization of a Diverse Panel of HIV-1 Env Pseudoviruses for Assessment of Neutralizing Antibodies

The restricted neutralization breadth of vaccine-elicited antibodies is a major limitation of current human immunodeficiency virus-1 (HIV-1) candidate vaccines. In order to permit the efficient identification of vaccines with enhanced capacity for eliciting cross-reactive neutralizing antibodies (NAbs) and to assess the overall breadth and potency of vaccine-elicited NAb reactivity, we assembled a panel of 109 molecularly cloned HIV-1 Env pseudoviruses representing a broad range of genetic and geographic diversity. Viral isolates from all major circulating genetic subtypes were included, as were viruses derived shortly after transmission and during the early and chronic stages of infection. We assembled a panel of genetically diverse HIV-1-positive (HIV-1⁺) plasma pools to assess the neutralization sensitivities of the entire virus panel. When the viruses were rank ordered according to the average sensitivity to neutralization by the HIV-1⁺ plasmas, a continuum of average sensitivity was observed. Clustering analysis of the patterns of sensitivity defined four subgroups of viruses: those having very high (tier 1A), above-average (tier 1B), moderate (tier 2), or low (tier 3) sensitivity to antibody-mediated neutralization. We also investigated potential associations between characteristics of the viral isolates (clade, stage of infection, and source of virus) and sensitivity to NAb. In particular, higher levels of NAb activity were observed when the virus and plasma pool were matched in clade. These data provide the first systematic assessment of the overall neutralization sensitivities of a genetically and geographically diverse panel of circulating HIV-1 strains. These reference viruses can facilitate the systematic characterization of NAb responses elicited by candidate vaccine immunogens.The development of an HIV-1 vaccine that can elicit protective humoral and cellular immunity is one of the highest priorities in the global fight against HIV/AIDS (2, 44). Data from lentiviral animal models suggest that antibodies capable of neutralizing primary strains of HIV-1 may have the capacity to prevent HIV-1 infection (1, 28, 30, 35). However, the ability to design immunogens that can elicit such broadly reactive neutralizing antibodies (NAbs) has proven to be a formidable obstacle, due in part to the extensive genetic diversity of HIV-1 and the complex escape mechanisms employed by the envelope gp120 and gp41 glycoproteins that form the trimeric viral envelope spike (Env) (20, 34, 45). As improved vaccine immunogens enter the stage of detailed preclinical analysis, the in vitro assays used for evaluating vaccine sera will need to detect incremental advances in the magnitude, breadth, and durability of NAb responses (37). Such data can then be used to distinguish and prioritize among antibody-based vaccine immunogens. Furthermore, highly reproducible and quantitative data on vaccine-elicited NAbs can enhance our understanding of the relationship between Env immunogen design and the resulting antibody response generated.Current recommendations for evaluating candidate vaccine sera for NAb activity include the use of standard reference panels of molecularly cloned HIV-1 Env pseudoviruses and a tiered algorithm of testing (27). Reference virus panels should represent genetically and geographically diverse subsets of viruses with neutralization phenotypes that are generally representative of primary isolate strains that a vaccine would need to protect against. As such, standard reference panels for HIV-1 subtypes B and C have been described (22, 23), and efforts continue toward the creation of virus reference panels representing additional genetic subtypes. For tiered evaluation of NAb activity, vaccine sera are first tested against homologous Env pseudoviruses and/or a small number of isolates that are known to be highly sensitive to antibody-mediated neutralization (commonly referred to as tier 1 viruses). A more rigorous assessment of the potency and breadth of vaccine-induced NAbs entails testing against more resistant reference panel viruses (commonly referred to as tier 2 viruses) that are either matched or mismatched in genetic subtype to the vaccine immunogen (second and third tiers of testing, respectively). This tiered approach for testing candidate HIV-1 vaccine sera is advantageous in that it provides increasingly stringent levels for assessing the potency and breadth of NAbs, uses standardized panels of reference viruses for consistency and reproducibility, and allows for the generation of comparative data sets for evaluating different candidate vaccine regimens.While the tiered algorithm for evaluating vaccine sera has gained acceptance in the field, a major limitation has been the lack of objective data to characterize HIV-1 Env pseudoviruses according to their overall sensitivity or resistance to antibody-mediated neutralization. The category of sensitive, tier 1 viruses arose in part from the observation that HIV-1 isolates passaged through T-cell lines often become highly sensitive to antibody-mediated neutralization (33). Compared to these laboratory-adapted viruses, most primary isolate strains are moderately resistant to NAbs. Yet, even among recently isolated circulating viral Envs, there is a wide spectrum of neutralization sensitivity. Some HIV-1 isolates have a neutralization phenotype closer to that of tier 1 viruses, while others appear to be quite neutralization resistant (6, 19, 22, 23). Overall, there are few data from which to understand or categorize the viral neutralization phenotypes of HIV-1 strains. As a result, we have a limited ability to assess the potential potency of vaccine-elicited NAbs or to estimate the percentage of circulating HIV-1 isolates that would be neutralized. Further categorization of isolates into distinct subgroups based on sensitivity to NAbs may reveal patterns of neutralization that could provide a greater understanding of the NAb response generated by current and future vaccine immunogens. In addition, the structure-based design of novel immunogens may be facilitated by an ability to monitor the types of viruses neutralized and to specifically map the viral epitopes targeted by vaccine-elicited NAbs.In this study, we assembled a diverse panel of 109 HIV-1 Env pseudoviruses, including multiple representatives from clades A, B, and C and circulating recombinant forms (CRFs) CRF07_BC and CRF02_AG-related. These were tested for their sensitivities using HIV-1-positive (HIV-1⁺) plasma samples representative of clades A, B, and C and CRF01_AE and CRF02_AG. Clinical, demographic, and viral genetic sequence data were collected for each virus. The neutralization phenotype of each virus was assessed with a panel of seven clade-specific HIV-1⁺ plasma pools. Viruses were rank ordered according to average neutralization sensitivity, and k-means clustering was utilized to identify four subgroups of viruses with neutralization phenotypes ranging from highly sensitive to resistant. Together, these results will improve the ability to rigorously evaluate antibody-based HIV-1 vaccines and will facilitate the interpretation of assay results to identify immunogens with improved capacity to elicit broadly cross-reactive NAbs.     

Profiling of Humoral Response to Influenza A(H1N1)pdm09 Infection and Vaccination Measured by a Protein Microarray in Persons with and without History of Seasonal Vaccination

Background

The influence of prior seasonal influenza vaccination on the antibody response produced by natural infection or vaccination is not well understood.

Methods

We compared the profiles of antibody responses of 32 naturally infected subjects and 98 subjects vaccinated with a 2009 influenza A(H1N1) monovalent MF59-adjuvanted vaccine (Focetria®, Novartis), with and without a history of seasonal influenza vaccination. Antibodies were measured by hemagglutination inhibition (HI) assay for influenza A(H1N1)pdm09 and by protein microarray (PA) using the HA1 subunit for seven recent and historic H1, H2 and H3 influenza viruses, and three avian influenza viruses. Serum samples for the infection group were taken at the moment of collection of the diagnostic sample, 10 days and 30 days after onset of influenza symptoms. For the vaccination group, samples were drawn at baseline, 3 weeks after the first vaccination and 5 weeks after the second vaccination.

Results

We showed that subjects with a history of seasonal vaccination generally exhibited higher baseline titers for the various HA1 antigens than subjects without a seasonal vaccination history. Infection and pandemic influenza vaccination responses in persons with a history of seasonal vaccination were skewed towards historic antigens.

Conclusions

Seasonal vaccination is of significant influence on the antibody response to subsequent infection and vaccination, and further research is needed to understand the effect of annual vaccination on protective immunity.     

Characterization of cross protection of Swine-Origin Influenza Virus (S-OIV) H1N1 and reassortant H5N1 influenza vaccine in BALB/c mice given a single-dose vaccination

Background

Influenza virus has antigen drift and antigen shift effect, vaccination with some influenza vaccine might not induce sufficient immunity for host to the threat of other influenza virus strains. S-OIV H1N1 and H5N1 influenza vaccines in single-dose immunization were evaluated in mice for cross protection to the challenge of A/California/7/2009 H1N1 or NIBRG-14 H5N1 virus.

Results

Both H1N1 and H5N1 induced significant homologous IgG, HAI, and microneutralization antibody responses in the mice, while only vaccines plus adjuvant produced significant heterogeneous IgG and HAI antibody responses. Both alum and MPLA adjuvants significantly reduced the S-OIV H1N1 vaccine dose required to elicit protective HAI antibody titers from 0.05 μg to 0.001 μg. Vaccines alone did not protect mice from challenge with heterogeneous influenza virus, while H5N1 vaccine plus alum and MPLA adjuvants did. Mouse body weight loss was also less significant in the presence of adjuvant than in the vaccine without adjuvant. Furthermore, both H1N1 and H5N1 lung viral titers of immunized mice were significantly reduced post challenge with homologous viruses.

Conclusion

Only in the presence of MPLA adjuvant could the H5N1 vaccine significantly reduce mouse lung viral titers post H1N1 virus challenge, and not vice versa. MPLA adjuvant induced cross protection with a single dose vaccination to the challenge of heterogeneous influenza virus in mice. Lung viral titer seemed to be a better indicator compared to IgG, neutralization antibody, and HAI titer to predict survival of mice infected with influenza virus.     

Stabilization of Influenza Vaccine Enhances Protection by Microneedle Delivery in the Mouse Skin

Background

Simple and effective vaccine administration is particularly important for annually recommended influenza vaccination. We hypothesized that vaccine delivery to the skin using a patch containing vaccine-coated microneedles could be an attractive approach to improve influenza vaccination compliance and efficacy.

Methodology/Principal Findings

Solid microneedle arrays coated with inactivated influenza vaccine were prepared for simple vaccine delivery to the skin. However, the stability of the influenza vaccine, as measured by hemagglutination activity, was found to be significantly damaged during microneedle coating. The addition of trehalose to the microneedle coating formulation retained hemagglutination activity, indicating stabilization of the coated influenza vaccine. For both intramuscular and microneedle skin immunization, delivery of un-stabilized vaccine yielded weaker protective immune responses including viral neutralizing antibodies, protective efficacies, and recall immune responses to influenza virus. Immunization using un-stabilized vaccine also shifted the pattern of antibody isotypes compared to the stabilized vaccine. Importantly, a single microneedle-based vaccination using stabilized influenza vaccine was found to be superior to intramuscular immunization in controlling virus replication as well as in inducing rapid recall immune responses post challenge.

Conclusions/Significance

The functional integrity of hemagglutinin is associated with inducing improved protective immunity against influenza. Simple microneedle influenza vaccination in the skin produced superior protection compared to conventional intramuscular immunization. This approach is likely to be applicable to other vaccines too.     

Japanese encephalitis virus replicon-based vaccine expressing enterovirus-71 epitope confers dual protection from lethal challenges

Background

To construct safer recombinant flavivirus vaccine, we exploited Japanese encephalitis virus (JEV) replicon-based platform to generate single-round infectious particles (SRIPs) that expressed heterologous neutralizing epitope SP70 derived from enterovirus-71 (EV71). Such pseudo-infectious virus particles, named SRIP-SP70, although are not genuine viable viruses, closely mimic live virus infection to elicit immune responses within one round of viral life cycle.

Results

We found that, besides gaining of full protection to thwart JEV lethal challenge, female outbred ICR mice, when were immunized with SRIP-SP70 by prime-boost protocol, could not only induce SP70-specific and IgG2a predominant antibodies but also provide their newborns certain degree of protection against EV71 lethal challenge.

Conclusions

Our results therefore exemplify that this vaccination strategy could indeed confer an immunized host a dual protective immunity against subsequent lethal challenge from JEV or EV71.