Infected Human Subjects Research Articles

Sensitivity and specificity of in vivo COVID-19 screening by detection dogs: Results of the C19-Screendog multicenter study

Trained dogs can recognize the volatile organic compounds contained in biological samples of patients with COVID-19 infection. We assessed the sensitivity and specificity of in vivo SARS-CoV-2 screening by trained dogs.We recruited five dog-handler dyads. In the operant conditioning phase, the dogs were taught to distinguish between positive and negative sweat samples collected from volunteers' underarms in polymeric tubes. The conditioning was validated by tests involving 16 positive and 48 negative samples held or worn in such a way that the samples were invisible to the dog and handler. In the screening phase the dogs were led by their handlers to a drive-through facility for in vivo screening of volunteers who had just received a nasopharyngeal swab from nursing staff. Each volunteer who had already swabbed was subsequently tested by two dogs, whose responses were recorded as positive, negative, or inconclusive. The dogs’ behavior was constantly monitored for attentiveness and wellbeing.All the dogs passed the conditioning phase, their responses showing a sensitivity of 83–100% and a specificity of 94–100%. The in vivo screening phase involved 1251 subjects, of whom 205 had a COVID-19 positive swab and two dogs per each subject to be screened. Screening sensitivity and specificity were respectively 91.6–97.6% and 96.3–100% when only one dog was involved, whereas combined screening by two dogs provided a higher sensitivity. Dog wellbeing was also analyzed: monitoring of stress and fatigue suggested that the screening activity did not adversely impact the dogs’ wellbeing. This work, by screening a large number of subjects, strengthen recent findings that trained dogs can discriminate between COVID-19 infected and healthy human subjects and introduce two novel research aspects: i) assessement of signs of fatigue and stress in dogs during training and testing, and ii) combining screening by two dogs to improve detection sensitivity and specificity.Using some precautions to reduce the risk of infection and spillover, in vivo COVID-19 screening by a dog-handler dyad can be suitable to quickly screen large numbers of people: it is rapid, non-invasive and economical, since it does not involve actual sampling, lab resources or waste management, and is suitable to screen large numbers of people.

Laboratory Culture and Life Cycle of Thelazia callipaeda in Intermediate and Definitive Hosts.

Human thelaziasis caused by Thelazia callipaeda is being increasingly reported worldwide. Notably, an epidemic trend is observed in Southwest China. Whether Phortica okadai found in Southwest China can act as a vector of T. callipaeda and human-derived T. callipaeda animal infections has not been widely reported. Here, P. okadai was maintained in a laboratory and experimentally infected with first-stage larvae collected from adult T. callipaeda that were isolated from infected human subjects. Dead P. okadai were subjected to PCR assay and dissected every two days to detect T. callipaeda. Subsequently, live flies were used to infect a rabbit. The infection procedures were performed once a day (20 min) for two weeks. The results show that L1 collected from the adult T. callipaeda could successfully parasitize P. okadai captured in Zunyi, a city in Southwest China, and developed into L3, and a rabbit was successfully infected with T. callipaeda using P. okadai as the intermediate host. The present study demonstrates a human-derived T. callipaeda infection in rabbits, through P. okadai, under laboratory conditions for the first time. These results provide insights into the transmission cycle of T. callipaeda and constitute a foundation to develop an effective treatment protocol for T. callipaeda infection.

Impact of Natural or Synthetic Singletons in the Capsid of Human Bocavirus 1 on Particle Infectivity and Immunoreactivity.

Human bocavirus 1 (HBoV1) is a parvovirus that gathers increasing attention due to its pleiotropic role as a pathogen and emerging vector for human gene therapy. Curiously, albeit a large variety of HBoV1 capsid variants has been isolated from human samples, only one has been studied as a gene transfer vector to date. Here, we analyzed a cohort of HBoV1-positive samples and managed to PCR amplify and sequence 29 distinct HBoV1 capsid variants. These differed from the originally reported HBoV1 reference strain in 32 nucleotides or four amino acids, including a frequent change of threonine to serine at position 590. Interestingly, this T590S mutation was associated with lower viral loads in infected patients. Analysis of the time course of infection in two patients for up to 15 weeks revealed a gradual accumulation of T590S, concurrent with drops in viral loads. Surprisingly, in a recombinant vector context, T590S was beneficial and significantly increased titers compared to that of T590 variants but had no major impact on their transduction ability or immunoreactivity. Additional targeted mutations in the HBoV1 capsid identified several residues that are critical for transduction, capsid assembly, or DNA packaging. Our new findings on the phylogeny, infectivity, and immunoreactivity of HBoV1 capsid variants improve our understanding of bocaviral biology and suggest strategies to enhance HBoV1 gene transfer vectors.IMPORTANCE The family of Parvoviridae comprises a wide variety of members that exhibit a unique biology and that are concurrently highly interesting as a scaffold for the development of human gene therapy vectors. A most notable example is human bocavirus 1 (HBoV1), which we and others have recently harnessed to cross-package and deliver recombinant genomes derived from another parvovirus, the adeno-associated virus (AAV). Here, we expanded the repertoire of known HBoV1 variants by cloning 29 distinct HBoV1 capsid sequences from primary human samples and by analyzing their properties as AAV/HBoV1 gene transfer vectors. This led to our discovery of a mutational hot spot at HBoV1 capsid position 590 that accumulated in two patients during natural infection and that lowers viral loads but increases vector yields. Thereby, our study expands our current understanding of HBoV1 biology in infected human subjects and concomitantly provides avenues to improve AAV/HBoV1 gene transfer vectors.

Transcriptomic approaches elucidate novel aspects of the host immune response to candidemia

Abstract Objective Examination of the human transcriptome in circulating leukocytes during invasive candidiasis can provide an innovative approach to defining immunologic mechanisms driving the host response to this deadly infection. Methods RNA sequencing was performed on peripheral blood samples from 50 hospitalized patients with blood culture positive invasive candidiasis, along with 30 control subjects. Generalized linear hypothesis testing and weighted gene co-expression network analysis (WGCNA) were used to define biological networks contributing to the host response to Candida infection. Results 1,748 genes were differentially upregulated and 2,149 genes downregulated in the presence of candidemia compared to matched controls. These genes reflected many canonical antifungal immune response pathways including phagocytosis, neutrophil activation, type I interferon responses, and T cell signaling. However, the data also demonstrated activation of some less well described antifungal immune mechanisms, including B cell activation, upregulation of humoral immune response pathways, and a profound alteration in the host regulation of heme synthesis and iron metabolism. While iron is known to be critical for fungal pathogens such as Candida in vitro, this analysis offers evidence that the human host manipulates this system as a part of the overall response to fungal infection. Conclusion Analysis of the transcriptome in circulating white blood cells of infected human subjects offers unique and novel insight into the complex immunologic response to invasive candidiasis.

In-depth proteomic characterization of Schistosoma haematobium: Towards the development of new tools for elimination.

BackgroundSchistosomiasis is a neglected disease affecting hundreds of millions worldwide. Of the three main species affecting humans, Schistosoma haematobium is the most common, and is the leading cause of urogenital schistosomiasis. S. haematobium infection can cause different urogenital clinical complications, particularly in the bladder, and furthermore, this parasite has been strongly linked with squamous cell carcinoma. A comprehensive analysis of the molecular composition of its different proteomes will contribute to developing new tools against this devastating disease.Methods and findingsBy combining a comprehensive protein fractionation approach consisting of OFFGEL electrophoresis with high-throughput mass spectrometry, we have performed the first in-depth characterisation of the different discrete proteomes of S. haematobium that are predicted to interact with human host tissues, including the secreted and tegumental proteomes of adult flukes and secreted and soluble egg proteomes. A total of 662, 239, 210 and 138 proteins were found in the adult tegument, adult secreted, soluble egg and secreted egg proteomes, respectively. In addition, we probed these distinct proteomes with urine to assess urinary antibody responses from naturally infected human subjects with different infection intensities, and identified adult fluke secreted and tegument extracts as being the best predictors of infection.ConclusionWe provide a comprehensive dataset of proteins from the adult and egg stages of S. haematobium and highlight their utility as diagnostic markers of infection intensity. Protein composition was markedly different between the different extracts, highlighting the distinct subsets of proteins that different development stages present in their different niches. Furthermore, we have identified adult fluke ES and tegument extracts as best predictors of infection using urine antibodies of naturally infected people. This study provides the first steps towards the development of novel tools to control this important neglected tropical disease.

Adaptive immune response in symptomatic and asymptomatic enteric protozoal infection: evidence for a determining role of parasite genetic heterogeneity in host immunity to human giardiasis

The genetic basis of the ultimate clinical outcomes of human giardiasis has been the subject of numerous investigations. We previously demonstrated roles for both host and parasite factors in determining the outcome of enteric infection in a murine model of Giardia duodenalis infection. In the current study, fecal and serum specimens from healthy controls and human subjects infected with the intestinal parasite G. duodenalis were assessed. Using a semi-nested PCR method, clinical isolates were genetically characterized based on the gdh and tpi loci, and the phylogenetic trees were constructed. Using a sandwich ELISA method, the serum levels of representative TH1 and TH2 cytokines were measured in infected human subjects and healthy controls. Here we showed that symptomatic human giardiasis was characterized by significantly elevated serum levels of the TH1 cytokine IFN-γ compared to healthy controls, whereas asymptomatic human subjects and healthy controls had comparable levels of serum IFN-γ. Further analyses showed that human subjects infected with G. duodenalis genotype AI had significantly elevated levels of serum IFN-γ and IL-10, but not IL-5, whereas human subjects infected with AII had similar levels of those cytokines compared to healthy controls. These data demonstrate roles for both host and parasite factors in the determination of the outcome of enteric infections and may further broaden our understanding of host-parasite interaction during enteric protozoal infections.

Achieving Potent Autologous Neutralizing Antibody Responses against Tier 2 HIV-1 Viruses by Strategic Selection of Envelope Immunogens

Advancement in immunogen selection and vaccine design that will rapidly elicit a protective Ab response is considered critical for HIV vaccine protective efficacy. Vaccine-elicited Ab responses must therefore have the capacity to prevent infection by neutralization-resistant phenotypes of transmitted/founder (T/F) viruses that establish infection in humans. Most vaccine candidates to date have been ineffective at generating Abs that neutralize T/F or early variants. In this study, we report that coimmunizing rhesus macaques with HIV-1 gp160 DNA and gp140 trimeric protein selected from native envelope gene sequences (envs) induced neutralizing Abs against Tier 2 autologous viruses expressing cognate envelope (Env). The Env immunogens were selected from envs emerging during the earliest stages of neutralization breadth developing within the first 2 years of infection in two clade B-infected human subjects. Moreover, the IgG responses in macaques emulated the targeting to specific regions of Env known to be associated with autologous and heterologous neutralizing Abs developed within the human subjects. Furthermore, we measured increasing affinity of macaque polyclonal IgG responses over the course of the immunization regimen that correlated with Tier 1 neutralization. In addition, we report firm correlations between Tier 2 autologous neutralization and Tier 1 heterologous neutralization, as well as overall TZM-bl breadth scores. Additionally, the activation of Env-specific follicular helper CD4 T cells in lymphocytes isolated from inguinal lymph nodes of vaccinated macaques correlated with Tier 2 autologous neutralization. These results demonstrate the potential for native Env derived from subjects at the time of neutralization broadening as effective HIV vaccine elements.

Infectomic Insights into the Roles of Exosymbiosis-Endosymbiosis Imbalance (EESI) in HIV-1 and SIV Infections

The vast majority of microbes form a healthy symbiotic ‘superorganism’ with the hosts. There are two types of symbiosis (Sym), exosymbiosis (e.g. microbiota) and endosymbiosis (e.g. mitochondria). It has been suggested that the exo-endo Sym balance (EESB) highly contribute to maintain the host homeostasis. However, alterations to the EESB caused by microbial (e.g. bacterial and viral pathogens) and non-microbial factors (e.g. substance abuse, diet and/or lifestyle) can disturb this symbiotic relationship and promote disease, such as inflammatory bowel diseases and acquired immune deficiency syndrome (AIDS). Progressive AIDS caused by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is characterized by systemic inflammation, opportunistic infection and malignant disorders resulting from generalized immune activation-mediated destruction of the healthy symbiotic super organism. Two extreme phenotypes are present in both HIV and SIV infections, including slow or rapid progression to AIDS (Pat: pathogenesis) in a majority of the infected human subjects and the non-natural primate host (i.e. rhesus macaques, RMs), and nonprogression to AIDS (Sym) in a minority of the infected people and the natural primate hosts (i.e. sooty mangabeys, SMs). Recently, it has been demonstrated that both exosymbiotic and endosymbiotic disorders contribute to the development of AIDS through infectomic studies of the extreme phenotypes of HIV/SIV infections. The involvement of the EESB in the pathogenesis and therapeutics of HIV infections is becoming increasingly clear. Indeed, many changes in the microbial diversity, abundance and composition of the gut microbiota and mitochondrial functions have been reported in HIV/AIDS, suggesting that there exist EESB problems in this disease. HIV virotoxins have been implicated in exploiting mitochondria to promote the targeted progressive and inexorable depletion of key immune cells (e.g. CD4 T cells), a hallmark of HIV/SIV infections. These findings support the notion that the exo-endo Sym imbalance (EESI) may play a central role in epidemiology, pathogenesis and management of infectious diseases, including AIDS caused by HIV-1 and SIV. Correction of the EESI problems in HIV/SIV infections may lead to a rational control of AIDS.

Th1-skewed tissue responses to a mycolyl glycolipid in mycobacteria-infected rhesus macaques

Trehalose 6,6′-dimycolate (TDM) is a major glycolipid of the cell wall of mycobacteria with remarkable adjuvant functions. To avoid detection by the host innate immune system, invading mycobacteria down-regulate the expression of TDM by utilizing host-derived glucose as a competitive substrate for their mycolyltransferases; however, this enzymatic reaction results in the concomitant biosynthesis of glucose monomycolate (GMM) which is recognized by the acquired immune system. GMM-specific, CD1-restricted T cell responses have been detected in the peripheral blood of infected human subjects and monkeys as well as in secondary lymphoid organs of small animals, such as guinea pigs and human CD1-transgenic mice. Nevertheless, it remains to be determined how tissues respond at the site where GMM is produced. Here we found that rhesus macaques vaccinated with Mycobacterium bovis bacillus Calmette–Guerin mounted a chemokine response in GMM-challenged skin that was favorable for recruiting T helper (Th)1 T cells. Indeed, the expression of interferon-γ, but not Th2 or Th17 cytokines, was prominent in the GMM-injected tissue. The GMM-elicited tissue response was also associated with the expression of monocyte/macrophage-attracting CC chemokines, such as CCL2, CCL4 and CCL8. Furthermore, the skin response to GMM involved the up-regulated expression of granulysin and perforin. Given that GMM is produced primarily by pathogenic mycobacteria proliferating within the host, the Th1-skewed tissue response to GMM may function efficiently at the site of infection.

Rapid, simple, and sensitive detection of the ompB gene of spotted fever group rickettsiae by loop-mediated isothermal amplification

BackgroundSpotted fever caused spotted fever group rickettsiae (SFGR) is prevalent throughout China. In this study, we describe a rapid, simple, and sensitive loop-mediated isothermal amplification (LAMP) assay targeting the ompB gene of spotted fever group rickettsiae ideal for application in China. The LAMP assay has the potential to detect spotted fever group rickettsiae early in infection and could therefore serve as an alternative to existing methods.MethodsA set of universal primers which are specific 7 common species of spotted fever group rickettsiae in China were designed using PrimerExplorer V4 software based on conserved sequences of ompB gene. The sensitivity, specificity and reproducibility of the LAMP were evaluated. The LAMP assay for detecting SFGR was compared with conventional PCR assays for sensitivity and specificity in early phase blood samples obtained from 11 infected human subjects.ResultsThe sensitivity of the LAMP assay was five copies per reaction (25 μL total volume), and the assay did not detect false-positive amplification across 42 strains of 27 members of the order Rickettsiales and 17 common clinical pathogens. The LAMP assay was negative to typhus group rickettsiae including R. prowazekii and R. typhi for no available conserved sequences of ompB was obtained for designing primers.To evaluate the clinical applicability of the LAMP assay, a total of 11 clinical samples, 10 samples confirmed serologically (3 cases), ecologically (1 case), by real-time polymerase chain reaction (PCR; 2 cases), ecologically and by real-time PCR (1 case), and serologically and by real-time PCR (3 cases) were analyzed by the ompB LAMP assay. Data were validated using a previously established nested PCR protocol and real-time PCR. A positive LAMP result was obtained for 8 of the 10 confirmed cases (sensitivity, 73%; specificity, 100%), while none of these samples were positive by nested PCR (sensitivity, 0%; specificity, 100%).ConclusionsThe LAMP assay described here is the most reliable among the three methods tested and would be an ideal choice for development as a rapid and cost-effective means of detecting SFGR in China.

Expansion of a subset of CD14highCD16negCCR2low/neg monocytes functionally similar to myeloid-derived suppressor cells during SIV and HIV infection

Monocytes have been categorized in three main subpopulations based on CD14 and CD16 surface expression. Classical monocytes express the CD14(++)CD16(-)CCR2(+) phenotype and migrate to inflammatory sites by quickly responding to CCL2 signaling. Here, we identified and characterized the expansion of a novel monocyte subset during HIV and SIV infection, which were undistinguishable from classical monocytes, based on CD14 and CD16 expression, but expressed significantly lower surface CCR2. Transcriptome analysis of sorted cells demonstrated that the CCR2(low/neg) cells are a distinct subpopulation and express lower levels of inflammatory cytokines and activation markers than their CCR2(high) counterparts. They exhibited impaired phagocytosis and greatly diminished chemotaxis in response to CCL2 and CCL7. In addition, these monocytes are refractory to SIV infection and suppress CD8(+) T cell proliferation in vitro. These cells express higher levels of STAT3 and NOS2, suggesting a phenotype similar to monocytic myeloid-derived cells, which suppress expansion of CD8(+) T cells in vivo. They may reflect an antiproliferative response against the extreme immune activation observed during HIV and SIV infections. In addition, they may suppress antiviral responses and thus, have a role in AIDS pathogenesis. Antiretroviral therapy in infected macaque and human subjects caused this population to decline, suggesting that this atypical phenotype is linked to viral replication.

Neutralization serotyping of BK polyomavirus infection in kidney transplant recipients.

BK polyomavirus (BKV or BKPyV) associated nephropathy affects up to 10% of kidney transplant recipients (KTRs). BKV isolates are categorized into four genotypes. It is currently unclear whether the four genotypes are also serotypes. To address this issue, we developed high-throughput serological assays based on antibody-mediated neutralization of BKV genotype I and IV reporter vectors (pseudoviruses). Neutralization-based testing of sera from mice immunized with BKV-I or BKV-IV virus-like particles (VLPs) or sera from naturally infected human subjects revealed that BKV-I specific serum antibodies are poorly neutralizing against BKV-IV and vice versa. The fact that BKV-I and BKV-IV are distinct serotypes was less evident in traditional VLP-based ELISAs. BKV-I and BKV-IV neutralization assays were used to examine BKV type-specific neutralizing antibody responses in KTRs at various time points after transplantation. At study entry, sera from 5% and 49% of KTRs showed no detectable neutralizing activity for BKV-I or BKV-IV neutralization, respectively. By one year after transplantation, all KTRs were neutralization seropositive for BKV-I, and 43% of the initially BKV-IV seronegative subjects showed evidence of acute seroconversion for BKV-IV neutralization. The results suggest a model in which BKV-IV-specific seroconversion reflects a de novo BKV-IV infection in KTRs who initially lack protective antibody responses capable of neutralizing genotype IV BKVs. If this model is correct, it suggests that pre-vaccinating prospective KTRs with a multivalent VLP-based vaccine against all BKV serotypes, or administration of BKV-neutralizing antibodies, might offer protection against graft loss or dysfunction due to BKV associated nephropathy.

The influence of RhD phenotype on toxoplasmosis- and age-associated changes in personality profile of blood donors

Toxoplasma gondii, a parasite infecting 20-60% of humans in various countries, influences the behaviour of infected animal and human hosts. Infected human subjects have changes in several of Cattell's and Cloninger's personality factors. Recently, three independent studies have shown that Rh-positive subjects are protected against the T. gondii-induced changes of reaction times and increased risk of traffic accidents. Here we searched for evidence of similar effects of RhD phenotype on toxoplasmosis- or aging-associated changes in the personality profile of about 302 blood donors. We found that Rh-positive and Rh-negative subjects responded differently to toxoplasmosis. In addition to the already known effects of toxoplasmosis on novelty seeking, self transcendence, superego strength and protension, we also found effects of RhD phenotype on ego strength, protension, and praxernia, as well as opposite effects of toxoplasmosis on ego strength, praxernia, ergic tension and cooperativeness in Rh-positive and Rh-negative subjects. Moreover, our results indicate that RhD phenotype might influence not only the effect of toxoplasmosis but also the effect of aging on specific personality traits.

Independent Evolution of Human Immunodeficiency Virus Type 1 env V1/V2 and V4/V5 Hypervariable Regions during Chronic Infection

Using DNA heteroduplex tracking assays, we characterized human immunodeficiency virus type 1 env V4/V5 genetic populations in multiple blood plasma samples collected over an average of 7 months from 24 chronically infected human subjects. We observed complex and dynamic V4/V5 genetic populations in most subjects. Comparisons of V4/V5 and V1/V2 population changes over the course of the study showed that major shifts in genetic populations frequently occurred in one region but not the other, and these observations were independently confirmed in one subject by single-genome sequencing. These results suggest that the V1/V2 and V4/V5 regions of env often evolve independently during chronic infection.

Correlation between Fundamental Binding Forces and Clinical Prognosis of Staphylococcus aureus Infections of Medical Implants

Atomic force microscopy was used to "fish" for binding reactions between a fibronectin-coated probe (i.e., substrate simulating an implant device) and each of 15 different isolates of Staphylococcus aureus obtained from either patients with an infected cardiac prosthesis (invasive group) or healthy human subjects (control group). There is a strong distinction (p = 0.01) in the binding-force signature observed for the invasive versus control populations. This observation suggests that a microorganism's "force taxonomy" may provide a fundamental and practical indicator of the pathogen-related risk that infections pose to patients with implanted medical devices.

Lopinavir‐Ritonavir: Effects on Endothelial Cell Function in Healthy Subjects

To differentiate between the effects that antiretroviral drugs have on the endothelium and the secondary effects that they have on immune function, viral load, and dyslipidemia, 6 non-human immunodeficiency virus-infected human subjects were treated with lopinavir-ritonavir for 1 month and, on the basis of forearm blood flow, the treatment's effects on endothelial cell function were measured. Surprisingly, after exposure to lopinavir-ritonavir, absolute forearm blood-flow responses to the endothelium-dependent vasodilator, acetylcholine, increased significantly (P=.03), and forearm blood flow decreased to a greater extent during specific inhibition of NO synthase by N(G)-monomethyl-L-arginine. Thus, in this small cohort of subjects, short-term treatment with lopinavir-ritonavir does not appear to directly promote endothelial cell dysfunction.

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection by duplex PCR assay based on telomeric sequences.

We used the species specificity and repetitious nature of subtelomeric kinetoplastida sequences to generate a duplex PCR assay for the simultaneous detection of Trypanosoma cruzi and Trypanosoma rangeli in experimentally and naturally infected triatomine (Reduviid) bugs and in infected human subjects. The assay was species specific and was capable of detecting 1/20th of T. cruzi and 1/4th of T. rangeli cell equivalents without complementary hybridization. In addition, the PCR-based assay was robust enough for direct application to difficult biological samples such as Reduviid feces or guts and was capable of recognizing all T. cruzi and T. rangeli strains and lineages. Because the assay primers amplify entirely different target sequences, no reaction interference was observed, facilitating future adaptation of this assay to an automated format.

Simultaneous determination of zidovudine and nevirapine in human plasma by RP-LC

A simple method for the simultaneous determination of zidovudine and nevirapine in human plasma by reversed-phase liquid chromatography with UV detection at 265 nm was developed. A solid–liquid extraction procedure with internal standard was applied to the samples prior to analysis. The system requires a Zorbax SB-C18 column, 250×4.6 mm I.D. and a mobile phase composed of potassium dihydrogen phosphate (10 mM; pH 6.5)-acetonitrile (83:17, v/v). Peak-areas are linear; correlation coefficients are better than 0.999; both inter- and intra-day accuracy and precision are lower than 15%. Extraction recoveries are higher than 90% for both zidovudine and nevirapine. The method proposed was employed to determine the levels of the two retroviral drugs in plasma from HIV infected human subjects.

Characterization of Haemophilus ducreyi-specific T-cell lines from lesions of experimentally infected human subjects.

Haemophilus ducreyi is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease that facilitates the transmission of human immunodeficiency virus. In the human model of infection, the histopathology of infected sites in part resembles a delayed-type hypersensitivity (DTH) response. In this study, T cells were isolated from skin biopsy specimens obtained from 24 subjects who were infected for 7 to 14 days. One clone and 12 lines that responded to H. ducreyi antigens were obtained from 12 of the subjects. Fluorescence-activated cell sorter analysis showed that the antigen-responsive lines and clone were predominantly CD3(+) and CD4(+). The lines and clone responded to H. ducreyi antigen in a dose-dependent manner and produced gamma interferon (IFN-gamma) alone or IFN-gamma and interleukin-10 (IL-10) but no IL-4 or IL-5 in response to H. ducreyi. Proliferation of T cells was dependent on the presence of autologous antigen-presenting cells. The lines showed little response to antigens prepared from other members of the Pasteurellaceae and responded to different fractions of H. ducreyi separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that T cells that recognize H. ducreyi antigens are recruited to sites experimentally infected with the organism. The lack of cross-reactivity to the Pasteurellaceae and the response of the lines to different antigen fractions suggest that subjects are sensitized to H. ducreyi during the course of infection.

Vaccination against Helicobacter pylori in non-human primate models and humans.

Several vaccination studies have been performed in monkeys and humans testing the feasibility of prophylactic and therapeutic immunizations against Helicobacter pylori. The monkey studies showed that immune responses were induced by oral vaccination with the mucosal adjuvant LT (Escherichia coli heat-labile enterotoxin), parenteral administration with a cationic lipid adjuvant, and by mucosal priming followed by parenteral boosts. Both prophylactic and therapeutic activities were demonstrated in monkeys, providing a strong impetus for human vaccine trials. Preliminary studies in humans were undertaken in order to identify a tolerable dose of LT adjuvant or to test the effectiveness of mutant atoxic LT adjuvants. The results from these preliminary studies suggest that native LT causes diarrhoea at doses required for adjuvanticity while a mutant LT does not. In one study in which infected human subjects were vaccinated with orally administered urease antigen with native LT, there was a modest reduction in the level of H. pylori gastric colonization. A second clinical study employing H. pylori whole cell antigen and a mutant LT in infected subjects showed immune responses and although the subjects remained infected, the study was not designed to measure reduction in H. pylori colonization. Recombinant Salmonella expressing urease and other H. pylori antigens have been effective in mice (see accompanying Frontlines Topic Review by John O. Nedrud [1]), but monkey studies are not possible because of host range restriction. Human trials of parenteral immunization, mucosal immunization with mutant LT and live Salmonella vectors are needed to fully assess the ability of vaccines to prevent or treat H. pylori infections.