Identification of the Feline Humoral Immune Response to Bartonella henselae Infection by Protein Microarray

Background

Bartonella henselae is the zoonotic agent of cat scratch disease and causes potentially fatal infections in immunocompromised patients. Understanding the complex interactions between the host''s immune system and bacterial pathogens is central to the field of infectious diseases and to the development of effective diagnostics and vaccines.

Methodology

We report the development of a microarray comprised of proteins expressed from 96% (1433/1493) of the predicted ORFs encoded by the genome of the zoonotic pathogen Bartonella henselae. The array was probed with a collection of 62 uninfected, 62 infected, and 8 “specific-pathogen free” naïve cat sera, to profile the antibody repertoire elicited during natural Bartonella henselae infection.

Conclusions

We found that 7.3% of the B. henselae proteins on the microarray were seroreactive and that seroreactivity was not evenly distributed between predicted protein function or subcellular localization. Membrane proteins were significantly most likely to be seroreactive, although only 23% of the membrane proteins were reactive. Conversely, we found that proteins involved in amino acid transport and metabolism were significantly underrepresented and did not contain any seroreactive antigens. Of all seroreactive antigens, 52 were differentially reactive with sera from infected cats, and 53 were equally reactive with sera from infected and uninfected cats. Thirteen of the seroreactive antigens were found to be differentially seroreactive between B. henselae type I and type II. Based on these results, we developed a classifier algorithm that was capable of accurately discerning 93% of the infected animals using the microarray platform. The seroreactivity and diagnostic potential of these antigens was then validated on an immunostrip platform, which correctly identified 98% of the infected cats. Our protein microarray platform provides a high-throughput, comprehensive analysis of the feline humoral immune response to natural infection with the alpha-proteobacterium B. henselae at an antigen-specific, sera-specific, and genome-wide level. Furthermore, these results provide novel insight and utility in diagnostics, vaccine development, and understanding of host-pathogen interaction.     

Recombinant Herpesvirus Glycoprotein G Improves the Protective Immune Response to Helicobacter pylori Vaccination in a Mouse Model of Disease

Alphaherpesviruses, which have co-evolved with their hosts for more than 200 million years, evade and subvert host immune responses, in part, by expression of immuno-modulatory molecules. Alphaherpesviruses express a single, broadly conserved chemokine decoy receptor, glycoprotein G (gG), which can bind multiple chemokine classes from multiple species, including human and mouse. Previously, we demonstrated that infection of chickens with an infectious laryngotracheitis virus (ILTV) mutant deficient in gG resulted in altered host immune responses compared to infection with wild-type virus. The ability of gG to disrupt the chemokine network has the potential to be used therapeutically. Here we investigated whether gG from ILTV or equine herpesvirus 1 (EHV-1) could modulate the protective immune response induced by the Helicobacter pylori vaccine antigen, catalase (KatA). Subcutaneous immunisation of mice with KatA together with EHV-1 gG, but not ILTV gG, induced significantly higher anti-KatA IgG than KatA alone. Importantly, subcutaneous or intranasal immunisation with KatA and EHV-1 gG both resulted in significantly lower colonization levels of H. pylori colonization following challenge, compared to mice vaccinated with KatA alone. Indeed, the lowest colonization levels were observed in mice vaccinated with KatA and EHV-1 gG, subcutaneously. In contrast, formulations containing ILTV gG did not affect H. pylori colonisation levels. The difference in efficacy between EHV-1 gG and ILTV gG may reflect the different spectrum of chemokines bound by the two proteins. Together, these data indicate that the immuno-modulatory properties of viral gGs could be harnessed for improving immune responses to vaccine antigens. Future studies should focus on the mechanism of action and whether gG may have other therapeutic applications.     

Prime-Boost Vaccination with Recombinant Mumps Virus and Recombinant Vesicular Stomatitis Virus Vectors Elicits an Enhanced Human Immunodeficiency Virus Type 1 Gag-Specific Cellular Immune Response in Rhesus Macaques

Intramuscular inoculation of rhesus macaques with one or more doses of recombinant vesicular stomatitis virus (rVSV) expressing human immunodeficiency virus type 1 (HIV-1) Gag (rVSVgag) typically elicits peak cellular immune responses of 500 to 1,000 gamma interferon (IFN-γ) enzyme-linked immunospots (ELISPOTS)/10⁶ peripheral blood lymphocytes (PBL). Here, we describe the generation of a novel recombinant mumps virus (rMuV) expressing HIV-1 Gag (rMuVgag) and measure the Gag-specific cellular immune responses detected in rhesus macaques following vaccination with a highly attenuated form of rVSV expressing HIV-1 Gag (rVSVN4CT1gag1) and rMuVgag in various prime-boost combinations. Notably, peak Gag-specific cellular immune responses of 3,000 to 3,500 ELISPOTS/10⁶ PBL were detected in macaques that were primed with rMuVgag and boosted with rVSVN4CT1gag1. Lower peak cellular immune responses were detected in macaques that were primed with rVSVN4CT1gag1 and boosted with rMuVgag, although longer-term gag-specific responses appeared to remain higher in this group of macaques. These findings indicate that rMuVgag may significantly enhance Gag-specific cellular immune responses when administered with rVSVN4CT1gag1 in heterologous prime-boost regimens.The ability to recover infectious virus from genomic cDNA has enabled the development of nonsegmented negative-strand RNA viruses as candidate vaccine vectors (8, 20); vesicular stomatitis virus (VSV), which predominantly infects insects and livestock in nature (29, 51, 52), is one of the most extensively studied in this group of RNA viruses. Recombinant forms of VSV (rVSVs) have been tested in preclinical studies as potential vaccine vectors to combat a wide range of human diseases including human immunodeficiency virus (HIV)/AIDS (16, 28, 30, 31, 40-43). In one of these studies, nonhuman primates (NHPs) vaccinated with rVSV vaccine vectors expressing simian immunodeficiency virus (SIV) Gag and HIV Env proteins were protected from disease following challenge with a pathogenic SIV/HIV-1 recombinant (SHIV) (46). Although these prototypic rVSV vaccine vectors elicited robust SHIV-specific immune responses in NHPs and demonstrated protective efficacy in the SHIV challenge model, they were found to be insufficiently attenuated for human trials when tested in a stringent NHP neurovirulence model (27). This finding was addressed by the development of a highly attenuated rVSV vector (rVSVN4CT1gag1). This vector was attenuated by combination of a specific N gene translocation and G gene truncation (9), with the N gene in position 4 (N4), the G gene expressing a G protein with a single amino acid in the cytoplasmic tail (CT1), and the HIV-1 Gag gene added in the first position of the genome (gag1). The rVSVN4CT1gag1 vector caused no obvious signs of neurological disease in young mice following intracranial inoculation with >10⁷ PFU of virus (12) and produced only very minimal, predominantly inflammatory lesions following intrathalamic inoculation of NHPs with 10⁷ PFU of virus (unpublished data). Although rVSVN4CT1gag1 demonstrated reduced in vitro replication efficiency and in vivo virulence, it was as immunogenic in mice (12) and NHPs (unpublished data) as the much more virulent prototypic rVSV vectors that provided protection from disease in the SHIV challenge model.Mumps virus (MuV), the agent of mumps in humans, is a nonsegmented negative-strand RNA virus in the family Paramyxoviridae. The incidence of mumps has been greatly reduced in the developed world by the introduction of live attenuated MuV vaccine strains over the past 30 to 35 years. The most commonly used MuV vaccine in the United States and Western Europe is the Jeryl Lynn strain, which has demonstrated excellent efficacy and an outstanding safety record for the >100 million doses administered to the pediatric population. A system for the recovery of the Jeryl Lynn strain of MuV from genomic cDNA has been described previously (10). This methodology has enabled targeted alteration of the MuV genome to study virus-associated neurovirulence and neuroattenuation (33) as well as the possibility of developing MuV as a vaccine vector for other pathogens.There is currently no proven method of inducing broadly neutralizing antibodies in HIV type 1 (HIV-1) vaccinees. It has been postulated, however, that robust vaccine-induced cellular immune responses directed against one or more HIV-1 proteins may be sufficient to prevent HIV-1-infected humans from developing AIDS in the absence of broadly neutralizing antibodies (34). Although this hypothesis has been called into question recently following the results of an HIV-1 phase II clinical trial (the STEP trial), there is still reason to believe that a robust cellular immune response against specific cytotoxic T-lymphocyte epitopes within highly conserved regions of the viral proteome could result in a significantly reduced viral load following HIV-1 infection (45). One rational approach for maximizing vaccine-induced HIV-1-specific peak cellular immune responses is the administration of completely heterologous vaccine vectors in prime-boost regimens (2, 15, 24, 40), unlike the serotype switch used previously in rVSV prime-boost vaccination regimens (12, 46, 47). In general, the magnitudes of the resulting cellular immune responses were higher than those detected for comparable prime-boost regimens with homologous vectors (14, 46) although any associated enhancement of protective efficacy in challenge models remains unclear (3, 48). Here, we describe the generation of a novel rMuV vector expressing HIV-1 Gag (rMuVgag) and the immune responses elicited in rhesus macaques when this vector was administered with a highly attenuated rVSVN4CT1gag1 vector in heterologous prime-boost regimens.     

Expanded Breadth of the T-Cell Response to Mosaic Human Immunodeficiency Virus Type 1 Envelope DNA Vaccination

An effective AIDS vaccine must control highly diverse circulating strains of human immunodeficiency virus type 1 (HIV-1). Among HIV-1 gene products, the envelope (Env) protein contains variable as well as conserved regions. In this report, an informatic approach to the design of T-cell vaccines directed to HIV-1 Env M group global sequences was tested. Synthetic Env antigens were designed to express mosaics that maximize the inclusion of common potential T-cell epitope (PTE) 9-mers and minimize the inclusion of rare epitopes likely to elicit strain-specific responses. DNA vaccines were evaluated using intracellular cytokine staining in inbred mice with a standardized panel of highly conserved 15-mer PTE peptides. One-, two-, and three-mosaic sets that increased theoretical epitope coverage were developed. The breadth and magnitude of T-cell immunity stimulated by these vaccines were compared to those for natural strain Envs; additional comparisons were performed on mutant Envs, including gp160 or gp145 with or without V regions and gp41 deletions. Among them, the two- or three-mosaic Env sets elicited the optimal CD4 and CD8 responses. These responses were most evident in CD8 T cells; the three-mosaic set elicited responses to an average of eight peptide pools, compared to two pools for a set of three natural Envs. Synthetic mosaic HIV-1 antigens can therefore induce T-cell responses with expanded breadth and may facilitate the development of effective T-cell-based HIV-1 vaccines.The development of AIDS vaccines has been advanced recently by demonstrations of increased survival and decreased viral load following vaccination with T-cell vaccines in nonhuman primate models (12, 19, 23, 26, 31, 37). Although such vaccine studies have implied that T cells may contribute to the control of viremia in the highly lethal simian immunodeficiency virus SIVmac251 challenge model, the applicability of these results in human studies remains uncertain. The major concern regarding the efficacy of human immunodeficiency virus (HIV) vaccines in humans is the extraordinary genetic diversity of the virus. The sequence similarity of HIV type 1 (HIV-1) envelope from diverse isolates within a clade can diverge by as much as 15%, and divergence between alternative clades may approach 30% (10). In addition, the diversity of the viral Gag gene product can approach similar levels, particularly in p17 and p15, which are much more diverse than p24 (6), although Gag does not have the extreme localized diversity seen in the highly variable regions of Env (6, 10). While the approach to viral diversity has been addressed in existing vaccines through the use of envelopes derived from representative viruses in the major clades, increasing knowledge about the genetic diversity of naturally occurring isolates has enabled alternative approaches that enhance population coverage of vaccine-elicited T-cell responses.Approaches under consideration include the use of central gene sequences based on ancestral, consensus, or center-of-the-tree genetic analyses (5, 10, 18, 31, 36). Such prototypes are derived by selection of the most common amino acids at each residue (10, 16, 17, 21, 25, 36), identifying the most recent common ancestor of diverging viruses in a vaccine target population (5, 10, 18, 36), or modeling the sequence at the center of the phylogenetic tree (29), respectively. Peptides based on any of these three centralized protein strategies enhanced the detection of T-cell responses in natural infection relative to the use of peptides based on natural strains; however, all three strategies behaved equivalently (7).The use of a single M group consensus/ancestral Env sequence has been shown to elicit T-cell responses with greater breadth of cross-reactivity than single natural strains in animal models (31, 36). Such central sequences do not exist in nature, and even phylogenetic ancestral reconstructions are just an approximate model of an ancestral state of the virus (8). Thus, central sequence strategies have provided evidence that various informatically derived gene products can elicit immune responses to T-cell epitopes found in diverse circulating strains, leading to the possibility of using computational strategies to design polyvalent vaccines which optimize T-cell coverage (6, 24). In this study, we have evaluated for the first time the ability of nonnatural mosaic Env immunogens (6) to elicit T-cell responses of increased cross-reactivity against epitopes represented in naturally circulating viruses in animals.Mosaic HIV-1 envelope genes were derived using an informatic approach, whereby in silico-generated recombinants of natural variants from the Los Alamos database M group Env alignment were created, scored, and selected in combination to optimize the coverage of 9-mers in the global database for a given vaccine cocktail size. While mosaic proteins are artificial constructs that do not occur in nature, they align well to natural proteins, and any short span found in mosaics will tend to be found repeatedly among natural strains (although some of the hypervariable loop regions of Env are so extremely variable that they are not repeated among circulating strains, and this necessitates bridging these regions with segments found in a single strain). In silico recombination breakpoints are constrained to create fusion points found in natural sequences. It is possible to provide increased breadth of coverage with a single mosaic, providing the maximum possible single-antigen diversity coverage for stretches of nine amino acids. Alternatively, multiple mosaics can increase the breadth of representation but have the drawback of requiring the synthesis of additional vectors for clinical use. Mosaics also preserve a natural Env-like sequence to retain normal antigen processing. Here, we have compared single-, double-, or triple-mosaic envelope antigen sets to naturally circulating strains or other derivatives for their ability to elicit immune responses of increased breadth. The data suggest that mosaic HIV-1 envelope sequences provide an approach that may be useful in the development of HIV vaccines that respond to T-cell epitopes represented in naturally circulating strains.     

Proteomics-Based Characterization of the Humoral Immune Response in Sporotrichosis: Toward Discovery of Potential Diagnostic and Vaccine Antigens

Background

Sporothrix schenckii and associated species are agents of human and animal sporotrichosis that cause large sapronoses and zoonoses worldwide. Epidemiological surveillance has highlighted an overwhelming occurrence of the highly pathogenic fungus Sporothrix brasiliensis during feline outbreaks, leading to massive transmissions to humans. Early diagnosis of feline sporotrichosis by demonstrating the presence of a surrogate marker of infection can have a key role for selecting appropriate disease control measures and minimizing zoonotic transmission to humans.

Methodology

We explored the presence and diversity of serum antibodies (IgG) specific against Sporothrix antigens in cats with sporotrichosis and evaluated the utility of these antibodies for serodiagnosis. Antigen profiling included protein extracts from the closest known relatives S. brasiliensis and S. schenckii. Enzyme-linked immunosorbent assays and immunoblotting enabled us to characterize the major antigens of feline sporotrichosis from sera from cats with sporotrichosis (n = 49), healthy cats (n = 19), and cats with other diseases (n = 20).

Principal Findings

Enzyme-linked immunosorbent assay-based quantitation of anti-Sporothrix IgG exhibited high sensitivity and specificity in cats with sporotrichosis (area under the curve, 1.0; 95% confidence interval, 0.94–1; P<0.0001) versus controls. The two sets of Sporothrix antigens were remarkably cross-reactive, supporting the hypothesis that antigenic epitopes may be conserved among closely related agents. One-dimensional immunoblotting indicated that 3-carboxymuconate cyclase (a 60-kDa protein in S. brasiliensis and a 70-kDa protein in S. schenckii) is the immunodominant antigen in feline sporotrichosis. Two-dimensional immunoblotting revealed six IgG-reactive isoforms of gp60 in the S. brasiliensis proteome, similar to the humoral response found in human sporotrichosis.

Conclusions

A convergent IgG-response in various hosts (mice, cats, and humans) has important implications for our understanding of the coevolution of Sporothrix and its warm-blooded hosts. We propose that 3-carboxymuconate cyclase has potential for the serological diagnosis of sporotrichosis and as target for the development of an effective multi-species vaccine against sporotrichosis in animals and humans.     

Highly Predictive Model for a Protective Immune Response to the A(H1N1)pdm2009 Influenza Strain after Seasonal Vaccination

Understanding the immune response after vaccination against new influenza strains is highly important in case of an imminent influenza pandemic and for optimization of seasonal vaccination strategies in high risk population groups, especially the elderly. Models predicting the best sero-conversion response among the three strains in the seasonal vaccine were recently suggested. However, these models use a large number of variables and/or information post- vaccination. Here in an exploratory pilot study, we analyzed the baseline immune status in young (<31 years, N = 17) versus elderly (≥50 years, N = 20) donors sero-negative to the newly emerged A(H1N1)pdm09 influenza virus strain and correlated it with the serological response to that specific strain after seasonal influenza vaccination. Extensive multi-chromatic FACS analysis (36 lymphocyte sub-populations measured) was used to quantitatively assess the cellular immune status before vaccination. We identified CD4⁺ T cells, and amongst them particularly naive CD4⁺ T cells, as the best correlates for a successful A(H1N1)pdm09 immune response. Moreover, the number of influenza strains a donor was sero-negative to at baseline (NSSN) in addition to age, as expected, were important predictive factors. Age, NSSN and CD4⁺ T cell count at baseline together predicted sero-protection (HAI≥40) to A(H1N1)pdm09 with a high accuracy of 89% (p-value = 0.00002). An additional validation study (N = 43 vaccinees sero-negative to A(H1N1)pdm09) has confirmed the predictive value of age, NSSN and baseline CD4⁺ counts (accuracy = 85%, p-value = 0.0000004). Furthermore, the inclusion of donors at ages 31–50 had shown that the age predictive function is not linear with age but rather a sigmoid with a midpoint at about 50 years. Using these results we suggest a clinically relevant prediction model that gives the probability for non-protection to A(H1N1)pdm09 influenza strain after seasonal multi-valent vaccination as a continuous function of age, NSSN and baseline CD4 count.     

壳聚糖介导增强DNA疫苗皮肤免疫效果的研究

目的:探讨壳聚糖(chitosan)包裹DNA疫苗皮肤免疫效果。方法:以chitosan包裹不同的草原兔尾鼠卵透明带3(LZP3)基因DNA疫苗pcDNA3-Lzp3(pLzp3)和pLC,通过皮肤擦拭免疫小鼠,以ELISA方法检测抗体水平,对卵巢做病理切片检测。结果:ELISA结果显示用chitosan包裹的pLC组在皮肤免疫中显示出优势,卵巢病理切片结果正常。结论:Chitosan包裹质粒皮肤免疫小鼠较纯质粒免疫效果更为显著。     

Acute Effects of Pathogenic Simian-Human Immunodeficiency Virus Challenge on Vaccine-Induced Cellular and Humoral Immune Responses to Gag in Rhesus Macaques

Simian-human immunodeficiency virus (SHIV) infection in macaques provides a convenient model for testing vaccine efficacy and for understanding viral pathogenesis in AIDS. We immunized macaques with recombinant, Salmonella typhimurium (expressing Gag) or soluble Gag in adjuvant to generate T-cell-dependent lymphoproliferative or serum antibody responses. Immunized animals were challenged by intrarectal inoculation with SHIV89.6PD. Virus infection was accompanied by rapid losses of lymphoproliferative responses to Gag or phytohemagglutinin. By 8 weeks, mitogen responses recovered to near normal levels but antigen-specific immunity remained at low or undetectable levels. Serum antibody levels were elevated initially by virus exposure but soon dropped well below levels achieved by immunization. Our studies show a rapid depletion of preexisting Gag-specific CD4⁺ T cells that prevent or limit subsequent antiviral cellular and humoral immune responses during acute SHIV infection.     

Protective Efficacy of a Single Immunization of a Chimeric Adenovirus Vector-Based Vaccine against Simian Immunodeficiency Virus Challenge in Rhesus Monkeys

Rare serotype and chimeric recombinant adenovirus (rAd) vectors that evade anti-Ad5 immunity are currently being evaluated as potential vaccine vectors for human immunodeficiency virus type 1 and other pathogens. We have recently reported that a heterologous rAd prime-boost regimen expressing simian immunodeficiency virus (SIV) Gag afforded durable partial immune control of an SIV challenge in rhesus monkeys. However, single-shot immunization may ultimately be preferable for global vaccine delivery. We therefore evaluated the immunogenicity and protective efficacy of a single immunization of chimeric rAd5 hexon hypervariable region 48 (rAd5HVR48) vectors expressing SIV Gag, Pol, Nef, and Env against a homologous SIV challenge in rhesus monkeys. Inclusion of Env resulted in improved control of peak and set point SIV RNA levels following challenge. In contrast, DNA vaccine priming did not further improve the protective efficacy of rAd5HVR48 vectors in this system.Heterologous prime-boost vaccine regimens have proven substantially more immunogenic than single vector immunizations in a variety of experimental models, but a single-shot vaccine would presumably be ideal for eventual global delivery. The potential utility of single-shot vaccines against pathogenic simian immunodeficiency virus (SIV) challenges in rhesus monkeys has not been well characterized. We therefore evaluated the protective efficacy of a single immunization of recombinant chimeric adenovirus type 5 (rAd5) hexon hypervariable region 48 (rAd5HVR48) vectors (15) expressing SIV Gag, Pol, Nef, and Env against a pathogenic SIV challenge in rhesus monkeys. These vectors contain the HVRs of the rare Ad48 serotype and have been shown to evade dominant Ad5 hexon-specific neutralizing antibodies (NAbs) (15). We also assessed the potential utility of inclusion of Env as an immunogen (6, 7, 17) and the degree to which DNA vaccine priming would enhance the protective efficacy afforded by a single rAd5HVR48 immunization (2, 7, 18, 21).Thirty adult rhesus monkeys (n = 6/group) lacking the Mamu-A*01, Mamu-B*17, and Mamu-B*08 class I alleles were primed with plasmid DNA vaccines and boosted with rAd5HVR48 vectors as follows: (1) adjuvanted DNA prime, rAd5HVR48 boost; (2) DNA prime, rAd5HVR48 boost; (3) rAd5HVR48 alone; (4) rAd5HVR48 alone (excluding Env); and (5) sham controls. Monkeys in groups 1 to 3 received vectors expressing SIVmac239 Gag, Pol, Nef, and Env, whereas monkeys in group 4 received vectors expressing only Gag, Pol, and Nef. The DNA vaccine adjuvants in group 1 were plasmids expressing the rhesus chemokine MIP-1α and Flt3L, which have been shown to increase recruitment of dendritic cells and to improve DNA vaccine immunogenicity (20). Monkeys were primed intramuscularly with a total dose of 4 mg of DNA vaccines at weeks 0, 4, and 8. All animals then received a single intramuscular immunization of 4 × 10¹⁰ viral particles (vp) of rAd5HVR48 at week 24. At week 52, animals were challenged intravenously (i.v.) with 100 monkey infectious doses of SIVmac251 (7, 10).     

Antibody-Dependent Transplacental Transfer of Malaria Blood-Stage Antigen Using a Human Ex Vivo Placental Perfusion Model

Prenatal exposure to allergens or antigens released by infections during pregnancy can stimulate an immune response or induce immunoregulatory networks in the fetus affecting susceptibility to infection and disease later in life. How antigen crosses from the maternal to fetal environment is poorly understood. One hypothesis is that transplacental antigen transfer occurs as immune complexes, via receptor-mediated transport across the syncytiotrophoblastic membrane and endothelium of vessels in fetal villi. This hypothesis has never been directly tested. Here we studied Plasmodium falciparum merozoite surface protein 1 (MSP1) that is released upon erythrocyte invasion. We found MSP1 in cord blood from a third of newborns of malaria-infected women and in >90% following treatment with acid dissociation demonstrating MSP1 immune complexes. Using an ex vivo human placental model that dually perfuses a placental cotyledon with independent maternal and fetal circuits, immune-complexed MSP1 transferred from maternal to fetal circulation. MSP1 alone or with non-immune plasma did not transfer; pre-incubation with human plasma containing anti-MSP1 was required. MSP1 bound to IgG was detected in the fetal perfusate. Laser scanning confocal microscopy demonstrated MSP1 in the fetal villous stroma, predominantly in fetal endothelial cells. MSP1 co-localized with IgG in endothelial cells, but not with placental macrophages. Thus we show, for the first time, antibody-dependent transplacental transfer of an antigen in the form of immune complexes. These studies imply frequent exposure of the fetus to certain antigens with implications for management of maternal infections during pregnancy and novel approaches to deliver vaccines or drugs to the fetus.     

Tropism of and Innate Immune Responses to the Novel Human Betacoronavirus Lineage C Virus in Human Ex Vivo Respiratory Organ Cultures

Since April 2012, there have been 17 laboratory-confirmed human cases of respiratory disease associated with newly recognized human betacoronavirus lineage C virus EMC (HCoV-EMC), and 7 of them were fatal. The transmissibility and pathogenesis of HCoV-EMC remain poorly understood, and elucidating its cellular tropism in human respiratory tissues will provide mechanistic insights into the key cellular targets for virus propagation and spread. We utilized ex vivo cultures of human bronchial and lung tissue specimens to investigate the tissue tropism and virus replication kinetics following experimental infection with HCoV-EMC compared with those following infection with human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome coronavirus (SARS-CoV). The innate immune responses elicited by HCoV-EMC were also investigated. HCoV-EMC productively replicated in human bronchial and lung ex vivo organ cultures. While SARS-CoV productively replicated in lung tissue, replication in human bronchial tissue was limited. Immunohistochemistry revealed that HCoV-EMC infected nonciliated bronchial epithelium, bronchiolar epithelial cells, alveolar epithelial cells, and endothelial cells. Transmission electron microscopy showed virions within the cytoplasm of bronchial epithelial cells and budding virions from alveolar epithelial cells (type II). In contrast, there was minimal HCoV-229E infection in these tissues. HCoV-EMC failed to elicit strong type I or III interferon (IFN) or proinflammatory innate immune responses in ex vivo respiratory tissue cultures. Treatment of human lung tissue ex vivo organ cultures with type I IFNs (alpha and beta IFNs) at 1 h postinfection reduced the replication of HCoV-EMC, suggesting a potential therapeutic use of IFNs for treatment of human infection.     

An Adjuvant for the Induction of Potent,Protective Humoral Responses to an H5N1 Influenza Virus Vaccine with Antigen-Sparing Effect in Mice

Intramuscular administration of inactivated influenza virus vaccine is the main vaccine platform used for the prevention of seasonal influenza virus infection. In clinical trials, inactivated H5N1 vaccines have been shown to be safe and capable of eliciting immune correlates of protection. However, the H5N1 vaccines are poorly immunogenic compared to seasonal influenza virus vaccines. Needle-free vaccination would be more efficient and economical in a pandemic, and the development of an effective and safe mucosal adjuvant will be an important milestone. A stabilized chemical analog of double-stranded RNA, PIKA, was previously reported to be a potent mucosal adjuvant in a murine model. While PIKA stimulates dendritic cells in vitro, little was known about its receptor and adjuvanting mechanism in vivo. In this study, we demonstrated that the immunostimulatory effect of PIKA resulted in an increased number of mature antigen-presenting cells, with the induction of proinflammatory cytokines at the inoculation site. In addition, coadministration of PIKA with a poorly immunogenic H5N1 subunit vaccine led to antigen sparing and quantitative and qualitative improvements of the immune responses over those achieved with an unadjuvanted vaccine in mice. The adjuvanted vaccine provided protection against lethal challenge with homologous and heterologous H5N1 wild-type viruses. Mice lacking functional TLR3 showed diminished cytokine production with PIKA stimulation, diminished antibody responses, and reduced protective efficacy against wild-type virus challenge following vaccination. These data suggest that TLR3 is important for the optimal performance of PIKA as an adjuvant. With its good safety profile and antigen-sparing effect, PIKA could be an attractive adjuvant for use in future pandemics.Influenza is an acute respiratory disease associated with significant morbidity and mortality worldwide. The newly emerged swine-origin H1N1 virus has caused the first influenza pandemic of this century (4). Since its appearance in April 2009, the virus has spread to every continent and caused significant morbidity and mortality (WHO website, http://gamapserver.who.int/h1n1/cases-deaths/h1n1_casesdeaths.html). The sporadic transmission of highly pathogenic avian influenza (HPAI) viruses (H5N1 influenza A viruses) from poultry to humans in Asia also raises concerns about a possible pandemic (2, 28).Although vaccination is the most effective tool for the control of influenza (7, 33), the combined production capacity of global vaccine suppliers is not sufficient to meet the demand during a pandemic, so a vaccine shortage is expected. Any strategy that can maximize vaccine coverage will be valuable in a pandemic.Inactivated seasonal influenza virus vaccines are administered mainly by the intramuscular (i.m.) route; however, it has been demonstrated that intranasal (i.n.) administration of inactivated influenza virus vaccines is more effective at inducing nasal IgA responses and protecting the respiratory epithelium (1, 47). Induction of immunity by the intranasal route often requires a high dose of vaccine or the inclusion of an adjuvant. Although a number of compounds have been identified as promising mucosal adjuvants, there is a need to continue to develop safe mucosal adjuvants, because some compounds, such as Escherichia coli heat-labile toxin and poly(I:C), are associated with significant side effects (27, 37).We previously demonstrated the potency of a stabilized chemical analog of double-stranded RNA (dsRNA), PIKA, as an adjuvant for a seasonal influenza virus vaccine with a substantial antigen-sparing effect in mice (25). While we and others have shown that PIKA activates dendritic cells (DC) in culture (25, 38), there are no reports on this effect in vivo, and the protective efficacy of PIKA-adjuvanted vaccine against wild-type (wt) virus challenge has not been demonstrated. The current study was designed to evaluate changes in the number and phenotypic expression of local antigen-presenting cells (APC) and in cytokine expression at the inoculation site and to evaluate the adjuvanting potency of PIKA in a lethal-challenge model using a wt influenza virus with pandemic potential. The A/Vietnam/1203/2004 (H5N1) virus was chosen over the A/California/04/2009 (H1N1) virus as the challenge virus for two reasons. First, the H5N1 virus is more virulent than the 2009 H1N1 pandemic virus in mice (the 50% mouse lethal doses [MLD₅₀] of the H5N1 and the H1N1 viruses are 10^0.4 and 10^5.8 50% tissue culture infective doses [TCID₅₀], respectively [20, 41]), which allows a higher lethal-challenge dose to be used in the experiments. Second, the unadjuvanted split-virion H5N1 vaccine was poorly immunogenic in humans, requiring 12 times more antigen (two doses of 90 μg) than the typical seasonal influenza virus vaccine (15 μg) in order to generate immunity associated with protection against influenza in humans (42), while data from the H1N1 human vaccine trial show that the unadjuvanted H1N1 vaccine is able to elicit robust immune responses after a single dose (14, 51). Our results show that administration of PIKA with inactivated H5N1 vaccine elicited a rapid production of proinflammatory cytokines with infiltration of mature DC at the site of administration. This vaccine formulation allowed significant antigen sparing and provided protection against lethal challenge with the wt HPAI viruses A/Vietnam/1203/2004 and A/Indonesia/05/2005 (H5N1).     

Improving the Safety of Mesenchymal Stem Cell-Based Ex Vivo Therapy Using Herpes Simplex Virus Thymidine Kinase

Human mesenchymal stem cells (MSCs) are multipotent stem cells that have been intensively studied as therapeutic tools for a variety of disorders. To enhance the efficacy of MSCs, therapeutic genes are introduced using retroviral and lentiviral vectors. However, serious adverse events (SAEs) such as tumorigenesis can be induced by insertional mutagenesis. We generated lentiviral vectors encoding the wild-type herpes simplex virus thymidine kinase (HSV-TK) gene and a gene containing a point mutation that results in an alanine to histidine substitution at residue 168 (TK(A168H)) and transduced expression in MSCs (MSC-TK and MSC-TK(A168H)). Transduction of lentiviral vectors encoding the TK(A168H) mutant did not alter the proliferation capacity, mesodermal differentiation potential, or surface antigenicity of MSCs. The MSC-TK(A168H) cells were genetically stable, as shown by karyotyping. MSC-TK(A168H) responded to ganciclovir (GCV) with an half maximal inhibitory concentration (IC₅₀) value 10-fold less than that of MSC-TK. Because MSC-TK(A168H) cells were found to be non-tumorigenic, a U87-TK(A168H) subcutaneous tumor was used as a SAE-like condition and we evaluated the effect of valganciclovir (vGCV), an oral prodrug for GCV. U87-TK(A168H) tumors were more efficiently ablated by 200 mg/kg vGCV than U87-TK tumors. These results indicate that MSC-TK(A168H) cells appear to be pre-clinically safe for therapeutic use. We propose that genetic modification with HSV-TK(A168H) makes allogeneic MSC-based ex vivo therapy safer by eliminating transplanted cells during SAEs such as uncontrolled cell proliferation.     

Immunoproteomics Analysis of the Murine Antibody Response to Vaccination with an Improved Francisella tularensis Live Vaccine Strain (LVS)

Background

Francisella tularensis subspecies tularensis is the causative agent of a spectrum of diseases collectively known as tularemia. An attenuated live vaccine strain (LVS) has been shown to be efficacious in humans, but safety concerns have prevented its licensure by the FDA. Recently, F. tularensis LVS has been produced under Current Good Manufacturing Practice (CGMP guidelines). Little is known about the immunogenicity of this new vaccine preparation in comparison with extensive studies conducted with laboratory passaged strains of LVS. Thus, the aim of the current work was to evaluate the repertoire of antibodies produced in mouse strains vaccinated with the new LVS vaccine preparation.

Methodology/Principal Findings

In the current study, we used an immunoproteomics approach to examine the repertoire of antibodies induced following successful immunization of BALB/c versus unsuccessful vaccination of C57BL/6 mice with the new preparation of F. tularensis LVS. Successful vaccination of BALB/c mice elicited antibodies to nine identified proteins that were not recognized by antisera from vaccinated but unprotected C57BL/6 mice. In addition, the CGMP formulation of LVS stimulated a greater repertoire of antibodies following vaccination compared to vaccination with laboratory passaged ATCC LVS strain. A total of 15 immunoreactive proteins were identified in both studies, however, 16 immunoreactive proteins were uniquely reactive with sera from the new formulation of LVS.

Conclusions/Significance

This is the first report characterising the antibody based immune response of the new formulation of LVS in the widely used murine model of tularemia. Using two mouse strains, we show that successfully vaccinated mice can be distinguished from unsuccessfully vaccinated mice based upon the repertoire of antibodies generated. This opens the door towards downselection of antigens for incorporation into tularemia subunit vaccines. In addition, this work also highlights differences in the humoral immune response to vaccination with the commonly used laboratory LVS strain and the new vaccine formulation of LVS.     

Temporal Analyses of Virus Replication, Immune Responses, and Efficacy in Rhesus Macaques Immunized with a Live, Attenuated Simian Immunodeficiency Virus Vaccine

Despite evidence that live, attenuated simian immunodeficiency virus (SIV) vaccines can elicit potent protection against pathogenic SIV infection, detailed information on the replication kinetics of attenuated SIV in vivo is lacking. In this study, we measured SIV RNA in the plasma of 16 adult rhesus macaques immunized with a live, attenuated strain of SIV (SIVmac239Δnef). To evaluate the relationship between replication of the vaccine virus and the onset of protection, four animals per group were challenged with pathogenic SIVmac251 at either 5, 10, 15, or 25 weeks after immunization. SIVmac239Δnef replicated efficiently in the immunized macaques in the first few weeks after inoculation. SIV RNA was detected in the plasma of all animals by day 7 after inoculation, and peak levels of viremia (10⁵ to 10⁷ RNA copies/ml) occurred by 7 to 12 days. Following challenge, SIVmac251 was detected in all of the four animals challenged at 5 weeks, in two of four challenged at 10 weeks, in none of four challenged at 15 weeks, and one of four challenged at 25 weeks. One animal immunized with SIVmac239Δnef and challenged at 10 weeks had evidence of disease progression in the absence of detectable SIVmac251. Although complete protection was not achieved at 5 weeks, a transient reduction in viremia (approximately 100-fold) occurred in the immunized macaques early after challenge compared to the nonimmunized controls. Two weeks after challenge, SIV RNA was also reduced in the lymph nodes of all immunized macaques compared with control animals. Taken together, these results indicate that host responses capable of reducing the viral load in plasma and lymph nodes were induced as early as 5 weeks after immunization with SIVmac239Δnef, while more potent protection developed between 10 and 15 weeks. In further experiments, we found that resistance to SIVmac251 infection did not correlate with the presence of antibodies to SIV gp130 and p27 antigens and was achieved in the absence of significant neutralizing activity against the primary SIVmac251 challenge stock.