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Background: Naja annulifera is a medically important venomous snake occurring in some of the countries in Sub-Saharan Africa. Accidental bites result in severe coagulation disturbances, systemic inflammation and heart damage, as reported in dogs, and death, by respiratory arrest, in humans. Despite the medical importance of N. annulifera, little is known about its venom composition and the pathogenesis of envenomation. In this paper, the toxic, inflammatory and immunogenic properties of N. annulifera venom were analyzed. Methodology/Principal findings: Venom proteomic analysis identified 79 different proteins, including Three Finger Toxins, Cysteine Rich Secretory Proteins, Metalloproteinases, Phospholipases A2 (PLA2), Hyaluronidase, L-amino-acid oxidase, Cobra Venom Factor and Serine Proteinase. The presence of PLA2, hyaluronidase, fibrinogenolytic and anticoagulant activities was detected using functional assays. The venom was cytotoxic to human keratinocytes. In an experimental murine model of envenomation, it was found that the venom induced local changes, such as swelling, which was controlled by anti-inflammatory drugs. Moreover, the venom caused death, which was preceded by systemic inflammation and pulmonary hemorrhage. The venom was shown to be immunogenic, inducing a strong humoral immune response, with the production of antibodies able to recognize venom components with high molecular weight and to neutralize its lethal activity. Conclusions/Significance: The results obtained in this study demonstrate that N. annulifera venom contains toxins able to induce local and systemic inflammation, which can contribute to lung damage and death. Moreover, the venom is immunogenic, an important feature that must be considered during the production of a therapeutic anti-N. annulifera antivenom. [ABSTRACT FROM AUTHOR]

With the HIV-1 epidemic in southern Africa still rising, a prophylactic vaccine against the region’s most prolific subtype (subtype C) would be a significant step forward. In this paper we report on the effect of 2 different adjuvants, AddaVax and AlhydroGel, formulated with HIV-1 subtype C gp140, on the development of binding and neutralising antibody titres in rabbits. AddaVax is a squalene-based oil-in-water nano-emulsion (similar to MF59) which can enhance both cellular and humoral immune responses, whilst AlhydroGel (aluminium hydroxide gel) mainly drives a Th2 response. The gp140 gene tested was derived from the superinfecting virus (SU) from participant CAP256 in the CAPRISA 002 Acute infection cohort. The furin cleavage site of the Env protein was replaced with a flexible linker and an I559P mutation introduced. Lectin affinity purified soluble Env protein was mainly trimeric as judged by molecular weight using BN-PAGE and contained intact broadly neutralising epitopes for the V3-glycan supersite (monoclonal antibodies PGT128 and PGT135), the CD4 binding site (VRC01) and the V2-glycan (PG9) but not for the trimer-specific monoclonal antibodies PG16, PGT145 and CAP256-VRC26_08. When this soluble Env protein was tested in rabbits, AlhydroGel significantly enhanced soluble Env and V1V2 binding antibodies when compared to AddaVax. Finally, AlhydroGel resulted in significantly higher neutralization titres for a subtype C Tier 1A virus (MW965.26) and increased neutralization breadth to Tier 1A and 1B viruses. However, no autologous Tier 2 neutralisation was observed. These data suggest that adjuvant selection is critical for developing a successful vaccine and AlhydroGel should be further investigated. Additional purification of trimeric native-like CAP256 Env and/or priming with DNA or MVA might enhance the induction of neutralizing antibodies and possible Tier 2 HIV-1 neutralisation. [ABSTRACT FROM AUTHOR]

Salmonella Typhi and Salmonella Paratyphi A are the leading causative agents of enteric fever which cause morbidity and mortality worldwide. Currently, there is no combination vaccine which could protect infection from both the strains. In this paper, we are focusing on the development of a novel bivalent typhoidal Outer Membrane Vesicles (OMVs) based immunogen against enteric fever. We have isolated Salmonella Typhi and Paratyphi A OMVs and also characterized OMVs associated antigens. Then we immunized adult mice with three doses of our newly formulated bivalent immunogen orally (25 μg/200 μl). After three doses of oral immunization, we found our immunogen could significantly induce humoral response. We have also found serum IgG against LPS, Vi-polysaccharide etc. OMV immunization induces CD4, CD8 and CD19 population in immunized mice spleen. It also induces Th1 and Th17-cell mediated immunity. We also found bivalent OMVs immunization can prevent more than lethal dose of heterologous Salmonella strains mediated systemic infection in adult mice model. We determined that, the protective immune responses depend on the humoral and cell-mediated immune response. Furthermore, we have evaluated the mode of protective immune response carried out by anti-OMVs antibody by significantly inhibiting bacterial motility and mucin penetration ability. Taken together, these findings suggest that our bivalent immunogen could be used as a novel candidate vaccine against enteric fever. [ABSTRACT FROM AUTHOR]

T-cells and antigen presenting cells are an essential part of the adaptive immune response system and how they interact is crucial in how the body effectively fights infection or responds to vaccines. Much of the experimental work studying interaction forces between cells has looked at the average properties of bulk samples of cells or applied microscopy to image the dynamic contact between these cells. In this paper we present a novel optical trapping technique for interrogating the force of this interaction and measuring relative interaction forces at the single-cell level. A triple-spot optical trap is used to directly manipulate the cells of interest without introducing foreign bodies such as beads to the system. The optical trap is used to directly control the initiation of cell-cell contact and, subsequently to terminate the interaction at a defined time point. The laser beam power required to separate immune cell pairs is determined and correlates with the force applied by the optical trap. As proof of concept, the antigen-specific increase in interaction force between a dendritic cell and a specific T-cell is demonstrated. Furthermore, it is demonstrated that this interaction force is completely abrogated when T-cell signalling is blocked. As a result the potential of using optical trapping to interrogate cellular interactions at the single cell level without the need to introduce foreign bodies such as beads is clearly demonstrated. [ABSTRACT FROM AUTHOR]

Marchuk's model of a general immune reaction is presented in the paper. The results of investigation of the model are summarized. The qualitative behaviour of solutions to the model and its simplification is described. Many illustrations of recovery process, oscillations or lethal outcomes of a disease are shown. The model with time-dependent immune reactivity is also considered. Periodic dynamics caused by different reasons are compared. In the last section adaptation of the model to tumour-immune interactions is proposed. The role of interleukins in immune processes is taken into account to adapt Marchuk's model to tumour growth dynamics. Possible immunotherapy is also considered in the proposed model. Some preliminary analysis of the model is presented. [ABSTRACT FROM AUTHOR]

There is increasing interest in multi-allele vaccines to overcome strain-specificity against polymorphic vaccine targets such as Apical Membrane Antigen 1 (AMA1). These have been shown to induce broad inhibitory antibodies in vitro and formed the basis for the design of three Diversity-Covering (DiCo) proteins with similar immunological effects. The antibodies produced are to epitopes that are shared between vaccine alleles and theoretically, increasing the number of component AMA1 alleles is expected to broaden the antibody response. A plateau effect could however impose a limit on the number of alleles needed to achieve the broadest specificity. Moreover, production cost and the vaccine formulation process would limit the number of component alleles. In this paper, we compare rabbit antibody responses elicited with multi-allele vaccines incorporating seven (three DiCos and four natural AMA1 alleles) and three (DiCo mix) antigens for gains in broadened specificity. We also investigate the effect of three adjuvant platforms on antigen specificity and antibody functionality. Our data confirms a broadened response after immunisation with DiCo mix in all three adjuvants. Higher antibody titres were elicited with either CoVaccine HTTM or Montanide ISA 51, resulting in similar in vitro inhibition (65-82%) of five out of six culture-adapted P. falciparum strains. The antigen binding specificities of elicited antibodies were also similar and independent of the adjuvant used or the number of vaccine component alleles. Thus neither the four extra antigens nor adjuvant had any observable benefits with respect to specificity broadening, although adjuvant choice influenced the absolute antibody levels and thus the extent of parasite inhibition. Our data confirms the feasibility and potential of multi-allele PfAMA1 formulations, and highlights the need for adjuvants with improved antibody potentiation properties for AMA1-based vaccines. [ABSTRACT FROM AUTHOR]

Abstract  Previous diagnosing methods based on agglutination have a limitation in view of emergency and point-of-care diagnoses due to the requirement of large scale equipments and much agglutination time. In this paper, we propose a low cost microfluidic lab-on-a-chip for more efficient detection of agglutination. In the present lab-on-a-chip, two inlet microwells, flow guiding microchannels, chaotic micromixer and reaction microwell are fully integrated. Mold inserts for the lab-on-a-chip were manufactured by UV photolithography and nickel electroplating process. The complete lab-on-a-chip was realized by the microinjection molding of cyclic olefin copolymer and the subsequent thermal bonding. The improved serpentine laminating micromixer, developed by our group, integrated in the lab-on-a-chip showed the high-level of chaotic mixing, thereby enabling us to get a reliable mixing of sample and reagent. The performance of the fabricated lab-on-a-chip was demonstrated by agglutination experiments with simulated bloods of 10 μl and simulated sera of 10 μl. The results of agglutination inside the reaction microwell were clearly read by means of the level of light transmission. The present microfluidic lab-on-a-chip could be widely applied to various clinical diagnostics based on agglutination tests. [ABSTRACT FROM AUTHOR]

This paper reviews the present knowledge of B-L antigens encoded by the chicken B complex as regards to the following aspects: (1) identification and cellular expression, (2) structural studies, (3) evidence for two distinct populations of B-L antigens, (4) mapping of B-L loci of the B complex, (5) B-L and immune response, and (6) the role of the B-L antigens for the control of mixed lymphocyte reactions (M LR) and graft-versus-host (GVH) reactions. It is concluded that B-L antigens of the chicken exhibit extensive homology with mammalian La antigens. A genetic map of the B complex is presented. [ABSTRACT FROM AUTHOR]

Immune responsiveness of guinea pigs to dinitrophenyl-poly-L-lysine and to the lysine rich random co-polymer of L-glutamic acid and L-lysine are both controlled by a single gene, the `poly-L-Iysine gene'. This paper reviews recent experiments which demonstrate that these two antigens specifically compete with one another for being presented to T cells by the same antigen-presenting cells. This finding is interpreted to mean that antigens to which responsiveness is controlled by the same single gene compete for the Ir gene product of antigen-presenting cells. The review discusses if the products of the immune response genes-presumably the Ia antigens-may constitute a third specific antigen recognition system. It further speculates if this idea may help to provide insight into the phenomenon of histocompatibility-restriction and into the nature of the mixed leucocyte reaction. [ABSTRACT FROM AUTHOR]

As perhaps the staunchest advocates of repertoire purging as the central mechanism of immunological tolerance, we note with satisfaction a spate of recent, elegant papers which suggest an intrathymic clonal abortion model as the explanation for at least some examples of T-cell tolerance. This view agrees with the classical formulation of the Billingham-Brent-Medawar school of tolerance as a specific, central failure of immune responsiveness. Repertoire purging within the B-lymphocyte compartment remains much more controversial. There is no doubt that experimental models exist where the B cell is the reversible target of tolerance induction. The question is, in view of the ease of inducing autoantibody formation both in vivo and in vitro, just how relevant are such clonal anergy mechanisms to authentic self-tolerance? Arguments are presented that there must be two windows of tolerance susceptibility in the ontogeny of the B cell; one while it is maturing in the bone marrow, to prevent autoreactivity of high affinity to important accessible self-antigens; and a second soon after activation of pre-memory cells by exogenous antigen, to prevent fortuitous mutations towards high-affinity anti-self-reactivity establishing a forbidden clone. [ABSTRACT FROM AUTHOR]

Although induced immunological tolerance of histocompatibility antigens is generally considered to be specific, cases of concomitant third-party unresponsiveness may occur. The present paper describes one case in point. Antigenic overlap between original and third-party donor, as well as runt disease have been excluded as possible explanations for the observed lack of specificity. It is hypothesized that an innate inability, of the host to respond to the relevant third-party antigen(s), or the ability of certain antigens to induce tolerance of themselves as well as of distinct but related antigens, may be responsible for the phenomenon. [ABSTRACT FROM AUTHOR]

The experiments described in this paper extend the observations of previous work in vitro demonstrating a decrease in the frequency of antigenreactive units specific for horse RBC in spleen cells from mice primed with KLH. It was observed that the addition of lymphoid cells having T-cell function reconstituted the anti-HRBC response to normal values. Significant enhancement of the response beyond the normal values could be evoked using two dissimilar methods, (i) addition of allogeneic thymus cells, and (ii) restimulation in vitro with the priming antigen. The latter type enhancement was elicited by the addition of small doses of KLH to cultures of spleen cells from mice recently primed with KLH, including cultures otherwise demonstrating antigen-induced suppression. The stimulus required to enhance the response to HRBC in these cultures was specific for the priming antigen, KLH. These results are discussed in the light of current theories of 'antigenic competition' and specific heterologous enhancement. [ABSTRACT FROM AUTHOR]

Anaplasma marginale is the most prevalent tick-borne livestock pathogen and poses a significant threat to cattle industry. In contrast to currently available live blood-derived vaccines against A. marginale, alternative safer and better-defined subunit vaccines will be of great significance. Two proteins (VirB9-1 and VirB9-2) from the Type IV secretion system of A. marginale have been shown to induce humoral and cellular immunity. In this study, Escherichia coli were used to express VirB9-1 and VirB9-2 proteins. Silica vesicles having a thin wall of 6 nm and pore size of 5.8 nm were used as the carrier and adjuvant to deliver these two antigens both as individual or mixed nano-formulations. High loading capacity was achieved for both proteins, and the mouse immunisation trial with individual as well as mixed nano-formulations showed high levels of antibody titres over 107 and strong T-cell responses. The mixed nano-formulation also stimulated high-level recall responses in bovine T-cell proliferation assays. These results open a promising path towards the development of efficient A. marginale vaccines and provide better understanding on the role of silica vesicles to deliver multivalent vaccines as mixed nano-formulations able to activate both B-cell and T-cell immunity, for improved animal health. [ABSTRACT FROM AUTHOR]

Sporotrichosis is a subcutaneous mycosis caused by a dimorphic complex of fungi called Sporothrix schenkii; it is a common disease in our country and has been reported in all continents. This paper presents a proposal of clinical classification of sporotrichosis based on its immunologic behavior, and which is divided into two groups, the first one hyperergic/normergic that presents a normal immune response to the intradermic antigen (sporotrichin M) and poor parasitic forms, and includes the most frequent clinical pictures: cutaneous-lymphatic and fixed-cutaneous; the second group or hypoergic/normergic, with low or no response to the intradermic antigen and presence of yeasts, includes the less frequent clinical pictures with opportunistic behavior: cutaneous-disseminated (hematogenous), cutaneous-superficial, osteoarticular, pulmonar and visceral. [ABSTRACT FROM AUTHOR]

We establish some properties of a within host mathematical model of malaria proposed by Recker et al. [M. Recker et al., Transient cross-reactive immune responses can orchestrate antigenic variation in malaria, Lett. Nature 429 (2004), pp. 555-558; M. Recker and S. Gupta, Conflicting immune responses can prolong the length of infection in Plasmodium falciparum malaria, Bull. Math. Biol. 68 (2006), pp. 821-835.], which includes the role of the immune system during the infection. The model accounts for the antigenic variation exhibited by the malaria parasite (Plasmodium falciparum). We show that the model can exhibit a wide variety of dynamical behaviours. We provide criteria for global stability, competitive exclusion and persistence. We also demonstrate that the disease equilibrium can be destabilized by non-symmetric cross-reactive responses. [ABSTRACT FROM AUTHOR]

This paper discusses some consequences of the discovery that antigen receptors are degenerate: Immune specificity, in contrast to the tenets of the clonal selection paradigm, must be generated by the immune response down-stream of initial antigen recognition; and specificity is a property of a collective of cells and not of single clones. [Copyright &y& Elsevier]

Rabbits made immunologically unresponsive by neonatal administration of HSA, HGG or BSA were given a course of intravenous injections of the respective antigens, adsorbed on alum, after a lapse of 13-27 months since the last administration of antigen. 8/12 responded to HSA, 4/5 to HGG, 9/10 to BSA, as judged by immune elimination of antigen, but this was delayed in onset and slow compared with that in previously untreated rabbits. The antibody formed was small in quantity and usually failed to precipitate with antigen. The sedimentation coefficients of 131I-labelled antigens, in the presence of excess antibody, were measured by ultracentrifugation through a sucrose density gradient. These showed that only small complexes were formed in some of the non-precipitating antisera. In one instance the diffusion coefficient of the complex was also measured, by a technique based on diffusion through agar gel. The calculated molecular weight of the complex, 330,000 indicated the presence of only two combining sites on the antigen. Combination of the anti-HSA sera with an HSA fragment was also measured. Whereas the amount of the fragment bound by ordinary hyperimmune anti-HSA sera was about one-fifth the HSA bound, the amounts bound by the test sera were relatively much less. Some non-precipitating sera failed to bind the fragment, although they bound HSA. These findings indicate that following neonatally induced immunological unresponsiveness the capacity to respond to antigen returns piecemeal in respect of different parts of the antigenic mosaic, and that it may be severely restricted. The theoretical implications are discussed. [ABSTRACT FROM AUTHOR]

The immunological responses of rabbits to HSA, HGG or BSA were tested at various times later in animals which had received the corresponding antigens before or shortly after birth. As judged by the criterion of failure to show immune elimination of antigen, a high proportion of the rabbits remained unresponsive at times when it was calculated that all the originally administered antigen would have been eliminated from the circulation. Furthermore, removal of antigen by passively administered antibody failed to restore the capacity to respond. It is concluded that, in respect of the antigens used, their persistence in the extracellular body fluids is not a prerequisite for maintenance of immunological unresponsiveness.
Further administration of the same antigen to rabbits which had escaped from a state of specific immunological unresponsiveness generally produced a very weak response, and in a few instances resulted in a return to the unresponsive state.
When the cross-reacting antigens HSA and BSA were administered adsorbed on alum to rabbits made unresponsive by neonatal contact with BSA and HSA respectively, and at the same time a further dose of the original antigen was given, antibodies were formed which were specific for the second antigen and did not cross-react with the first. In only 1/9 animals was responsiveness to the first antigen restored. The significance of these results is discussed. [ABSTRACT FROM AUTHOR]

A state of immunological paralysis has been induced in adult CBA mice by intrapertioneal injections of small quantities of bovine γ globulin (BGG). The minimum paralyzing dose of BGG has been found to be between 50 and 200 μg. A dose as small as 2 μg, has been found to have a slight paralyzing effect. The time necessary for the induction of paralysis by 50 μg to 2 mg of BGG in CBA mice is 3-4 days. Paralysis is induced by only one component of BGG; this component is incapable of inducing an antibody response unless an injection of adjuvant is made at the same time or slightly before the injection of the antigen. The BGG is centrifuged at an RCF of 20,00-30,000 g to remove particulate matter. Failure to remove the particulate matter leads to sporadic immune responses in groups of mice injected with the protein. Mice given a paralyzing injection of BGG were subsequently challenged by an injection of BGG in Freund's adjuvant. The result of this challenge was tested by an injection of radioactively-labelled antigen and the elimination of this antigen from the circulation of the challenged mice was followed for several days. 'Immune elimination' can easily be distinguished from 'non-immune elimination'. The presence of antibody to the non-paralysing components of BGG in sera from paralysed mice was confirmed using the Ouchterlony gel-diffusion technique. [ABSTRACT FROM AUTHOR]

Rabbit spleen cells, which have been treated in vitro with certain rabbit or guinea-pig antisera (e.g. against human serum albumin) and washed, are capable of specifically adsorbing radio-isotope labelled antigen. The antibody responsible for this effect appears to be distinct from the main precipitating anti-body in the serum and the term ‘cytophilic antibody’ has been suggested. The activity of the cytophilic antibody is not destroyed by heating at 56° C. for half an hour. The cytophilic antibody is not adsorbed by red cells. Spleen cells which have been treated with methanol before treatment with antiserum take up less antigen than non-methanol-treated cells tested in the same way. However, the addition of flesh normal spleen cells to the methanol-antibody-treated cells before the addition of antigen increased the uptake of the latter. [ABSTRACT FROM AUTHOR]

We analysed data from 80 patients who tested positive for SARS‐CoV‐2 RNA who had previously been HLA typed to support transplantation. Data were combined from two adjacent centres in Manchester and Leeds to achieve a sufficient number for early analysis. HLA frequencies observed were compared against two control populations: first, against published frequencies in a UK deceased donor population (n = 10,000) representing the target population of the virus, and second, using a cohort of individuals from the combined transplant waiting lists of both centres (n = 308), representing a comparator group of unaffected individuals of the same demographic. We report a significant HLA association with HLA‐ DQB1*06 (53% vs. 36%; p <.012; OR 1.96; 95% CI 1.94–3.22) and infection. A bias towards an increased representation of HLA‐A*26, HLA‐DRB1*15, HLA‐DRB1*10 and DRB1*11 was also noted but these were either only significant using the UK donor controls, or did not remain significant after correction for multiple tests. Likewise, HLA‐A*02, HLA‐B*44 and HLA‐C*05 may exert a protective effect, but these associations did not remain significant after correction for multiple tests. This is relevant information for the clinical management of patients in the setting of the current SARS‐CoV‐2 pandemic and potentially in risk‐assessing staff interactions with infected patients. [ABSTRACT FROM AUTHOR]

Background: Plasmodium malariae is the third most prevalent human malaria-causing species and has a patchy, but ample distribution in the world. Humans can host the parasite for years without presenting significant symptoms, turning its diagnosis and control into a difficult task. Here, we investigated the immunogenicity of recombinant proteins of P. malariae MSP1. Methods: Five regions of PmMSP1 were expressed in Escherichia coli as GST-fusion proteins and immunized in BALB/c mice. The specificity, subtyping, and affinity of raised antibodies were evaluated by enzyme-linked immunosorbent assays. Cellular immune responses were analyzed by lymphoproliferation assays and cytokine levels produced by splenocytes were detected by cytometry. Results: We found that N-terminal, central regions, and PmMSP119 are strongly immunogenic in mice. After three doses, the induced immune responses remained high for 70 days. While antibodies induced after immunization with N-terminal and central regions showed similar affinities to the target antigens, affinities of IgG against PmMSP119 were higher. All proteins induced similar antibody subclass patterns (predominantly IgG1, IgG2a, and IgG2b), characterizing a mixed Th1/Th2 response. Further, autologous stimulation of splenocytes from immunized mice led to the secretion of IL2 and IL4, independently of the antigen used. Importantly, IgG from P. malariae-exposed individuals reacted against PmMSP1 recombinant proteins with a high specificity. On the other hand, sera from P. vivax or P. falciparum-infected individuals did not react at all against recombinant PmMSP1 proteins. Conclusion: Recombinant PmMSP1 proteins are very useful diagnostic markers of P. malariae in epidemiological studies or in the differential diagnosis of malaria caused by this species. Immunization with recombinant PmMSP1 proteins resulted in a significant humoral immune response, which may turn them potential component candidates for a vaccine against P. malariae. [ABSTRACT FROM AUTHOR]

This open-label study assessed the safety and immunogenicity of two doses (14 days apart) of an indigenously manufactured, killed, bivalent (Vibrio cholerae O1 and O139), whole-cell oral cholera vaccine (SHANCHOL; Shantha Biotechnics) in healthy adults (n = 100) and children (n = 100) in a cholera endemic area (Vellore, South India) to fulfill post-licensure regulatory requirements and post-World Health Organization (WHO) prequalification commitments. Safety and reactogenicity were assessed, and seroconversion rates (i.e. proportion of participants with a ≥ 4-fold rise from baseline in serum vibriocidal antibody titers against V. cholerae O1 Inaba, O1 Ogawa and O139, respectively) were determined 14 days after each vaccine dose. No serious adverse events were reported during the study. Commonly reported solicited adverse events were headache and general ill feeling. Seroconversion rates after the first and second dose in adults were 67.7% and 55.2%, respectively, against O1 Inaba; 47.9% and 45.8% against O1 Ogawa; and 19.8% and 20.8% against O139. In children, seroconversion rates after the first and second dose were 80.2% and 68.8%, respectively, against O1 Inaba; 72.9% and 67.7% against O1 Ogawa; and 26.0% and 18.8% against O139. The geometric mean titers against O1 Inaba, O1 Ogawa, and O139 in both adults and children were significantly higher after each vaccine dose compared to baseline titers (P < 0.001; for both age groups after each dose versus baseline). The seroconversion rates for O1 Inaba, O1 Ogawa, and O139 in both age groups were similar to those in previous studies with the vaccine. In conclusion, the killed, bivalent, whole-cell oral cholera vaccine has a good safety and reactogenicity profile, and is immunogenic in healthy adults and children. Trial Registration: ClinicalTrials.gov ; CTRI/2012/01/002354. [ABSTRACT FROM AUTHOR]

To permit the recognition of antigens, T cells generate a vast diversity of T cell receptor (TCR) sequences. Upon binding of the TCR to an antigen-MHC complex, T cells clonally expand to establish an immune response. To study antigen-specific T cell clonality, we have developed a method that allows selection of rare cells, based on RNA expression, before in-depth scRNA-seq (named SELECT-seq). We applied SELECT-seq to collect both TCR sequences and then transcriptomes from single cells of peripheral blood lymphocytes activated by a Mycobacterium tuberculosis (Mtb) lysate. TCR sequence analysis allowed us to preferentially select expanded conventional CD8+ T cells as well as invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells. The iNKT and MAIT cells have a highly similar transcriptional pattern, indicating that they carry out similar immunological functions and differ considerably from conventional CD8+ T cells. While there is no relationship between expression profiles and clonal expansion in iNKT or MAIT cells, highly expanded conventional CD8+ T cells down-regulate the interleukin 2 (IL-2) receptor alpha (IL2RA, or CD25) protein and show signs of senescence. This suggests inherent limits to clonal expansion that act to diversify the T cell response repertoire. [ABSTRACT FROM AUTHOR]

The vaccine elicitation of broadly neutralizing responses is a central goal of HIV research. Recently, we elicited cross-clade neutralizing responses against the N terminus of the fusion peptide (FP), a critical component of the HIV-entry machinery. While the consistency of the elicited cross-clade neutralizing responses was good in mice, it was poor in guinea pigs: after seven immunizations comprising either envelope (Env) trimer or FP coupled to a carrier, serum from only one of five animals could neutralize a majority of a cross-clade panel of 19 wild-type strains. Such a low response rate—only 20%—made increasing consistency an imperative. Here, we show that additional Env-trimer immunizations could boost broad FP-directed neutralizing responses in a majority of immunized animals. The first boost involved a heterologous Env trimer developed from the transmitted founder clade C strain of donor CH505, and the second boost involved a cocktail that combined the CH505 trimer with a trimer from the BG505 strain. After boosting, sera from three of five animals neutralized a majority of the 19-strain panel and serum from a fourth animal neutralized 8 strains. We demonstrate that cross-reactive serum neutralization targeted the FP by blocking neutralization with soluble fusion peptide. The FP competition revealed two categories of elicited responses: an autologous response to the BG505 strain of high potency (~10,000 ID50), which was not competed by soluble FP, and a heterologous response of lower potency, which was competed by soluble FP. While the autologous response could increase rapidly in response to Env-trimer boost, the heterologous neutralizing response increased more slowly. Overall, repetitive Env-trimer immunizations appeared to boost low titer FP-carrier primed responses to detectable levels, yielding cross-clade neutralization. The consistent trimer-boosted neutralizing responses described here add to accumulating evidence for the vaccine utility of the FP site of HIV vulnerability. [ABSTRACT FROM AUTHOR]

Necrotic enteritis is an economically important poultry disease caused by the bacterium Clostridium perfringens. There are currently no necrotic enteritis vaccines commercially available for use in broiler birds, the most important target population. Salmonella-vectored vaccines represent a convenient and effective option for controlling this disease. We used a single attenuated Salmonella vaccine strain, engineered to lyse within the host, to deliver up to three C. perfringens antigens. Two of the antigens were toxoids, based on C. perfringens α-toxin and NetB toxin. The third antigen was fructose-1,6-bisphosphate aldolase (Fba), a metabolic enzyme with an unknown role in virulence. Oral immunization with a single Salmonella vaccine strain producing either Fba, α-toxoid and NetB toxoid, or all three antigens, was immunogenic, inducing serum, cellular and mucosal responses against Salmonella and the vectored C. perfringens antigens. All three vaccine strains were partially protective against virulent C. perfringens challenge. The strains delivering Fba only or all three antigens provided the best protection. We also demonstrate that both toxins and Fba are present on the C. perfringens cell surface. The presence of Fba on the cell surface suggests that Fba may function as an adhesin. [ABSTRACT FROM AUTHOR]

Plasmodium vivax remains a global health problem and its ability to cause relapses and subpatent infections challenge control and elimination strategies. Even in low malaria transmission settings, such as the Amazon basin, where progress in malaria control has caused a remarkable reduction in case incidence, a recent increase in P. vivax transmission demonstrates the continued vulnerability of P.vivax-exposed populations. As part of a search for complementary approaches to P.vivax surveillance in areas in which adults are the majority of the exposed-population, here we evaluated the potential of serological markers covering a wide range of immunogenicity to estimate malaria transmission trends. For this, antibodies against leading P. vivax blood-stage vaccine candidates were assessed during a 9 year follow-up study among adults exposed to unstable malaria transmission in the Amazon rainforest. Circulating antibody levels against immunogenic P. vivax proteins, such as the Apical Membrane Antigen-1, were a sensitive measure of recent P. vivax exposure, while antibodies against less immunogenic proteins were indicative of naturally-acquired immunity, including the novel engineered Duffy binding protein II immunogen (DEKnull-2). Our results suggest that the robustness of serology to estimate trends in P.vivax malaria transmission will depend on the immunological background of the study population, and that for adult populations exposed to unstable P.vivax malaria transmission, the local heterogeneity of antibody responses should be considered when considering use of serological surveillance. [ABSTRACT FROM AUTHOR]

Francisella tularensis is an intracellular pathogen causing the disease tularemia, and an organism of concern to biodefence. There is no licensed vaccine available. Subunit approaches have failed to induce protection, which requires both humoral and cellular immune memory responses, and have been hampered by a lack of understanding as to which antigens are immunoprotective. We undertook a preliminary in silico analysis to identify candidate protein antigens. These antigens were then recombinantly expressed and encapsulated into glucan particles (GPs), purified Saccharomyces cerevisiae cell walls composed primarily of β-1,3-glucans. Immunological profiling in the mouse was used to down-selection to seven lead antigens: FTT1043 (Mip), IglC, FTT0814, FTT0438, FTT0071 (GltA), FTT0289, FTT0890 (PilA) prior to transitioning their evaluation to a Fischer 344 rat model for efficacy evaluation. F344 rats were vaccinated with the GP protein antigens co-delivered with GP-loaded with Francisella LPS. Measurement of cell mediated immune responses and computational epitope analysis allowed down-selection to three promising candidates: FTT0438, FTT1043 and FTT0814. Of these, a GP vaccine delivering Francisella LPS and the FTT0814 protein was able to induce protection in rats against an aerosol challenge of F. tularensis SchuS4, and reduced organ colonisation and clinical signs below that which immunisation with a GP-LPS alone vaccine provided. This is the first report of a protein supplementing protection induced by LPS in a Francisella vaccine. This paves the way for developing an effective, safe subunit vaccine for the prevention of inhalational tularemia, and validates the GP platform for vaccine delivery where complex immune responses are required for prevention of infections by intracellular pathogens. [ABSTRACT FROM AUTHOR]

Human herpesviruses are antigenically rich agents that induce strong CD8+T cell responses in primary infection yet persist for life, continually challenging T cell memory through recurrent lytic replication and potentially influencing the spectrum of antigen-specific responses. Here we describe the first lytic proteome-wide analysis of CD8+ T cell responses to a gamma1-herpesvirus, Epstein-Barr virus (EBV), and the first such proteome-wide analysis of primary versus memory CD8+ T cell responses to any human herpesvirus. Primary effector preparations were generated directly from activated CD8+ T cells in the blood of infectious mononucleosis (IM) patients by in vitro mitogenic expansion. For memory preparations, EBV-specific cells in the blood of long-term virus carriers were first re-stimulated in vitro by autologous dendritic cells loaded with a lysate of lytically-infected cells, then expanded as for IM cells. Preparations from 7 donors of each type were screened against each of 70 EBV lytic cycle proteins in combination with the donor’s individual HLA class I alleles. Multiple reactivities against immediate early (IE), early (E) and late (L) lytic cycle proteins, including many hitherto unrecognised targets, were detected in both contexts. Interestingly however, the two donor cohorts showed a different balance between IE, E and L reactivities. Primary responses targeted IE and a small group of E proteins preferentially, seemingly in line with their better presentation on the infected cell surface before later-expressed viral evasins take full hold. By contrast, target choice equilibrates in virus carriage with responses to key IE and E antigens still present but with responses to a select subset of L proteins now often prominent. We infer that, for EBV at least, long-term virus carriage with its low level virus replication and lytic antigen release is associated with a re-shaping of the virus-specific response. [ABSTRACT FROM AUTHOR]

Determining the duration of protective immunity requires quantifying the magnitude and rate of loss of antibodies to different virus and vaccine antigens. A key complication is heterogeneity in both the magnitude and decay rate of responses of different individuals to a given vaccine, as well as of a given individual to different vaccines. We analyzed longitudinal data on antibody titers in 45 individuals to characterize the extent of this heterogeneity and used models to determine how it affected the longevity of protective immunity to measles, rubella, vaccinia, tetanus, and diphtheria. Our analysis showed that the magnitude of responses in different individuals varied between 12- and 200-fold (95% coverage) depending on the antigen. Heterogeneity in the magnitude and decay rate contribute comparably to variation in the longevity of protective immunity between different individuals. We found that some individuals have, on average, slightly longer-lasting memory than others—on average, they have higher antibody levels with slower decay rates. We identified different patterns for the loss of protective levels of antibodies to different vaccine and virus antigens. Specifically, we found that for the first 25 to 50 years, virtually all individuals have protective antibody titers against diphtheria and tetanus, respectively, but about 10% of the population subsequently lose protective immunity per decade. In contrast, at the outset, not all individuals had protective titers against measles, rubella, and vaccinia. However, these antibody titers wane much more slowly, with a loss of protective immunity in only 1% to 3% of the population per decade. Our results highlight the importance of long-term longitudinal studies for estimating the duration of protective immunity and suggest both how vaccines might be improved and how boosting schedules might be reevaluated. [ABSTRACT FROM AUTHOR]

Background: Plasmodium knowlesi is the most common cause of malaria in Malaysian Borneo, with reporting limited to clinical cases presenting to health facilities and scarce data on the true extent of transmission. Serological estimations of transmission have been used with other malaria species to garner information about epidemiological patterns. However, there are a distinct lack of suitable serosurveillance tools for this neglected disease. Methodology/Principal findings: Using in silico tools, we designed and expressed four novel P. knowlesi protein products to address the distinct lack of suitable serosurveillance tools: PkSERA3 antigens 1 and 2, PkSSP2/TRAP and PkTSERA2 antigen 1. Antibody prevalence to these antigens was determined by ELISA for three time-points post-treatment from a hospital-based clinical treatment trial in Sabah, East Malaysia (n = 97 individuals; 241 total samples for all time points). Higher responses were observed for the PkSERA3 antigen 2 (67%, 65/97) across all time-points (day 0: 36.9% 34/92; day 7: 63.8% 46/72; day 28: 58.4% 45/77) with significant differences between the clinical cases and controls (n = 55, mean plus 3 SD) (day 0 p<0.0001; day 7 p<0.0001; day 28 p<0.0001). Using boosted regression trees, we developed models to classify P. knowlesi exposure (cross-validated AUC 88.9%; IQR 86.1–91.3%) and identified the most predictive antibody responses. Conclusions/Significance: The PkSERA3 antigen 2 had the highest relative variable importance in all models. Further validation of these antigens is underway to determine the specificity of these tools in the context of multi-species infections at the population level. [ABSTRACT FROM AUTHOR]

As part of a major project to investigate protective and diagnostic immune markers against tuberculosis (TB), we measured antibody isotype responses to Mycobacterium tuberculosis (Mtb) antigens (LAM, Rv2031, and HBHA) in cohorts of 149 pulmonary tuberculosis patients (PTBP), 148 household contacts (HHCs), and 68 community controls (CCs) in an endemic setting. ELISA was used to measure levels of IgA, IgG, and IgM from sera of cohorts at baseline, and at 6 and 12 months from entry. The results show that there were significant differences in IgA, IgG, and IgM responses to the different antigens and in the three cohorts. At baseline, the level of IgM against RV2031 and LAM did not vary between cohorts, but the levels of IgA and IgG against Rv2031 were significantly higher in PTB patients than HHCs and CCs, followed by HHCs, and the lowest in CCs. In patients, there was a significant variation in antibody responses before and after chemotherapy. The levels of IgA and IgG against HBHA, and IgA against Rv2031 decreased significantly and remained low, while IgA and IgG against LAM increased significantly and remained high following chemotherapy. However, the levels of IgM against Rv2031 and LAM increased at 6 months but decreased again at 12 months. IgM against HBHA did not show any significant variation before and after chemotherapy. Similarly, there were also significant variations in antibody responses in HHCs over time. Our results show that there are significant variations in IgA, IgG and IgM responses to the different antigens and in the three cohorts, implying that not all antibody isotype responses are markers of clinical TB. In addition, the current and previous studies consistently show that IgA and IgG against Rv2031 discriminate between clinical disease, Mtb-infected and non-infected individuals. [ABSTRACT FROM AUTHOR]

Campylobacter is the leading cause of human bacterial gastroenteritis in the European Union. Birds represent the main reservoir of the bacteria, and human campylobacteriosis mainly occurs after consuming and/or handling poultry meat. Reducing avian intestinal Campylobacter loads should impact the incidence of human diseases. At the primary production level, several measures have been identified to reach this goal, including vaccination of poultry. Despite many studies, however, no efficient vaccine is currently available. We have recently identified new vaccine candidates using the reverse vaccinology strategy. This study assessed the in vivo immune and protective potential of six newly-identified vaccine antigens. Among the candidates tested on Ross broiler chickens, four (YP_001000437.1, YP_001000562.1, YP_999817.1, and YP_999838.1) significantly reduced cecal Campylobacter loads by between 2 and 4.2 log10 CFU/g, with the concomitant development of a specific humoral immune response. In a second trial, cecal load reductions results were not statistically confirmed despite the induction of a strong immune response. These vaccine candidates need to be further investigated since they present promising features. [ABSTRACT FROM AUTHOR]

This study aims to characterize the antigenicity of the Capsid (C) protein and the human antibody responses to C protein from the four dengue virus (DENV) serotypes. Parker hydrophilicity prediction, Emini surface accessibility prediction and Karplus & Schulz flexibility predictions were used to bioinformatically characterize antigenicity. The human antibody response to C protein was assessed by ELISA using immune sera and an array of overlapping DENV2 C peptides. DENV2 C protein peptides P1 (located on C protein at 2–18 a.a), P11 (79–95 a.a) and P12 (86–101 a.a) were recognized by most individuals exposed to infections with only one of the 4 DENV serotypes as well as people exposed to infections with two serotypes. These conserved peptide epitopes are located on the amino (1–40 a.a) and carboxy (70–100 a.a) terminal regions of C protein, which were predicted to be antigenic using different bioinformatic tools. DENV2 C peptide P6 (39–56 a.a) was recognized by all individuals exposed to DENV2 infections, some individuals exposed to DENV4 infections and none of the individuals exposed to DENV1 or 3 infections. Thus, unlike C peptides P1, P11 and P12, which contain epitopes, recognized by DENV serotype cross-reactive antibodies, DENV2 peptide P6 contains an epitope that is preferentially recognized by antibodies in people exposed to this serotype compared to other serotypes. We discuss our results in the context of the known structure of C protein and recent work on the human B-cell response to DENV infection. [ABSTRACT FROM AUTHOR]

Many biopharmaceuticals (BPs) are known to be immunogenic in the clinic, which can result in modified pharmacokinetics, reduced efficacy, allergic reactions and anaphylaxis. During recent years, several technologies to predict immunogenicity have been introduced, but the predictive value is still considered low. Thus, there is an unmet medical need for optimization of such technologies. The generation of T cell dependent high affinity anti-drug antibodies plays a key role in clinical immunogenicity. This study aimed at developing and evaluating a novel in vitro T cell:PBMC assay for prediction of the immunogenicity potential of BPs. To this end, we assessed the ability of infliximab (anti-TNF-α), rituximab (anti-CD20), adalimumab (anti-TNF-α) and natalizumab (anti-α4-integrin), all showing immunogenicity in the clinic, to induce a CD4+ T cells response. Keyhole limpet hemocyanin (KLH) and cytomegalovirus pp65 protein (CMV) were included as neo-antigen and recall antigen positive controls, respectively. By analyzing 26 healthy donors having HLA-DRB1 alleles matching the European population, we calculated the frequency of responding donors, the magnitude of the response, and the frequency of BP-specific T cells, as measured by 3[H]-thymidine incorporation and ELISpot IL-2 secretion. KLH and CMV demonstrated a strong T cell response in all the donors analyzed. The frequency of responding donors to the BPs was 4% for infliximab, 8% for adalimumab, 19% for rituximab and 27% for natalizumab, which is compared to and discussed with their respective observed clinical immunogenicity. This study further complements predictive immunogenicity testing by quantifying the in vitro CD4+ T cell responses to different BPs. Even though the data generated using this modified method does not directly translate to the clinical situation, a high sensitivity and immunogenic potential of most BPs is demonstrated. [ABSTRACT FROM AUTHOR]

Introduction: Helminth infection may affect vaccine immunogenicity and efficacy. Adolescents, a target population for tuberculosis booster vaccines, often have a high helminth burden. We investigated effects of Schistosoma mansoni (Sm) on the immunogenicity and safety of MVA85A, a model candidate tuberculosis vaccine, in BCG-vaccinated Ugandan adolescents. Methods: In this phase II open label trial we enrolled 36 healthy, previously BCG-vaccinated adolescents, 18 with no helminth infection detected, 18 with Sm only. The primary outcome was immunogenicity measured by Ag85A-specific interferon gamma ELISpot assay. Tuberculosis and schistosome-specific responses were also assessed by whole-blood stimulation and multiplex cytokine assay, and by antibody ELISAs. Results: Ag85A-specific cellular responses increased significantly following immunisation but with no differences between the two groups. Sm infection was associated with higher pre-immunisation Ag85A-specific IgG4 but with no change in antibody levels following immunisation. There were no serious adverse events. Most reactogenicity events were of mild or moderate severity and resolved quickly. Conclusions: The significant Ag85A-specific T cell responses and lack of difference between Sm-infected and uninfected participants is encouraging for tuberculosis vaccine development. The implications of pre-existing Ag85A-specific IgG4 antibodies for protective immunity against tuberculosis among those infected with Sm are not known. MVA85A was safe in this population. Trial registration: ClinicalTrials.gov [ABSTRACT FROM AUTHOR]

Background: Schistosomiasis, a severe disease caused by parasites of the genus Schistosoma, is prevalent in 74 countries, affecting more than 250 million people, particularly children. We have previously shown that the Schistosoma mansoni gut-derived cysteine peptidase, cathepsin B1 (SmCB1), administered without adjuvant, elicits protection (>60%) against challenge infection of S. mansoni or S. haematobium in outbred, CD-1 mice. Here we compare the immunogenicity and protective potential of another gut-derived cysteine peptidase, S. mansoni cathepsin L3 (SmCL3), alone, and in combination with SmCB1. We also examined whether protective responses could be boosted by including a third non-peptidase schistosome secreted molecule, glyceraldehyde 3-phosphate dehydrogenase (SG3PDH), with the two peptidases. Methodology/Principal findings: While adjuvant-free SmCB1 and SmCL3 induced type 2 polarized responses in CD-1 outbred mice those elicited by SmCL3 were far weaker than those induced by SmCB1. Nevertheless, both cysteine peptidases evoked highly significant (P < 0.005) reduction in challenge worm burden (54–65%) as well as worm egg counts and viability. A combination of SmCL3 and SmCB1 did not induce significantly stronger immune responses or higher protection than that achieved using each peptidase alone. However, when the two peptidases were combined with SG3PDH the levels of protection against challenge S. mansoni infection reached 70–76% and were accompanied by highly significant (P < 0.005) decreases in worm egg counts and viability. Similarly, high levels of protection were achieved in hamsters immunized with the cysteine peptidase/SG3PDH-based vaccine. Conclusions/Significance: Gut-derived cysteine peptidases are highly protective against schistosome challenge infection when administered subcutaneously without adjuvant to outbred CD-1 mice and hamsters, and can also act to enhance the efficacy of other schistosome antigens, such as SG3PDH. This cysteine peptidase-based vaccine should now be advanced to experiments in non-human primates and, if shown promise, progressed to Phase 1 safety trials in humans. [ABSTRACT FROM AUTHOR]

In an effort to make affordable vaccines suitable for the regions most affected by HIV-1, we have constructed stable vaccines that express an HIV-1 subtype C mosaic Gag immunogen (BCG-GagM, MVA-GagM and DNA-GagM). Mosaic immunogens have been designed to address the tremendous diversity of this virus. Here we have shown that GagM buds from cells infected and transfected with MVA-GagM and DNA-GagM respectively and forms virus-like particles. Previously we showed that a BCG-GagM prime MVA-GagM boost generated strong cellular immune responses in mice. In this study immune responses to the DNA-GagM and MVA-GagM vaccines were evaluated in homologous and heterologous prime-boost vaccinations. The DNA homologous prime boost vaccination elicited predominantly CD8+ T cells while the homologous MVA vaccination induced predominantly CD4+ T cells. A heterologous DNA-GagM prime MVA-GagM boost induced strong, more balanced Gag CD8+ and CD4+ T cell responses and that were predominantly of an effector memory phenotype. The immunogenicity of the mosaic Gag (GagM) was compared to a naturally occurring subtype C Gag (GagN) using a DNA homologous vaccination regimen. DNA-GagN expresses a natural Gag with a sequence that was closest to the consensus sequence of subtype C viruses sampled in South Africa. DNA-GagM homologous vaccination induced cumulative HIV-1 Gag-specific IFN-γ ELISPOT responses that were 6.5-fold higher than those induced by the DNA-GagN vaccination. Similarly, DNA-GagM vaccination generated 7-fold higher levels of cytokine-positive CD8+ T cells than DNA-GagN, indicating that this subtype C mosaic Gag elicits far more potent immune responses than a consensus-type Gag. Cells transfected and infected with DNA-GagM and MVA-GagM respectively, expressed high levels of GagM and produced budding virus-like particles. Our data indicates that a heterologous prime boost regimen using DNA and MVA vaccines expressing HIV-1 subtype C mosaic Gag is highly immunogenic in mice and warrants further investigation in non-human primates. [ABSTRACT FROM AUTHOR]

Background: Leprosy reactions, reversal reactions/RR and erythema nodosum leprosum/ENL, can cause irreversible nerve damage, handicaps and deformities. The study of Mycobacterium leprae-specific serologic responses at diagnosis in the cohort of patients enrolled at the Clinical Trial for Uniform Multidrug Therapy Regimen for Leprosy Patients in Brazil/U-MDT/CT-BR is suitable to evaluate its prognostic value for the development of reactions. Methodology: IgM and IgG antibody responses to PGL-I, LID-1, ND-O-LID were evaluated by ELISA in 452 reaction-free leprosy patients at diagnosis, enrolled and monitored for the development of leprosy reactions during a total person-time of 780,930 person-days, i.e. 2139.5 person-years, with a maximum of 6.66 years follow-up time. Principal findings: Among these patients, 36% (160/452) developed reactions during follow-up: 26% (119/452) RR and 10% (41/452) had ENL. At baseline higher anti-PGL-I, anti-LID-1 and anti-ND-O-LID seropositivity rates were seen in patients who developed ENL and RR compared to reaction-free patients (p<0.0001). Seroreactivity in reactional and reaction-free patients was stratified by bacilloscopic index/BI categories. Among BI negative patients, higher anti-PGL-I levels were seen in RR compared to reaction-free patients (p = 0.014). In patients with 0

While influenza virus diversity and antigenic drift have been well characterized on a global scale, the factors that influence the virus’ rapid evolution within and between human hosts are less clear. Given the modest effectiveness of seasonal vaccination, vaccine-induced antibody responses could serve as a potent selective pressure for novel influenza variants at the individual or community level. We used next generation sequencing of patient-derived viruses from a randomized, placebo-controlled trial of vaccine efficacy to characterize the diversity of influenza A virus and to define the impact of vaccine-induced immunity on within-host populations. Importantly, this study design allowed us to isolate the impact of vaccination while still studying natural infection. We used pre-season hemagglutination inhibition and neuraminidase inhibition titers to quantify vaccine-induced immunity directly and to assess its impact on intrahost populations. We identified 166 cases of H3N2 influenza over 3 seasons and 5119 person-years. We obtained whole genome sequence data for 119 samples and used a stringent and empirically validated analysis pipeline to identify intrahost single nucleotide variants at ≥1% frequency. Phylogenetic analysis of consensus hemagglutinin and neuraminidase sequences showed no stratification by pre-season HAI and NAI titer, respectively. In our study population, we found that the vast majority of intrahost single nucleotide variants were rare and that very few were found in more than one individual. Most samples had fewer than 15 single nucleotide variants across the entire genome, and the level of diversity did not significantly vary with day of sampling, vaccination status, or pre-season antibody titer. Contrary to what has been suggested in experimental systems, our data indicate that seasonal influenza vaccination has little impact on intrahost diversity in natural infection and that vaccine-induced immunity may be only a minor contributor to antigenic drift at local scales. [ABSTRACT FROM AUTHOR]

HIV-1 infection is associated with an early and profound depletion of mucosal memory CD4+ T cells, a population that plays an indispensable role in the regulation of isotype switching and transepithelial transport of antibodies. In this study, we addressed whether the depletion of CD4+ T cell in HIV-1-infected individuals results in altered humoral responses specific to antigens encountered at mucosal surfaces. Comprehensive protein microarray of systemic humoral responses to intestinal microbiota demonstrated reduced IgG responses to antigens derived from Proteobacteria and Firmicutes but not Bacteroidetes. Importantly, intestinal secretions of antiretroviral therapy-treated HIV-1-infected individuals exhibited a significant elevation of IgM levels and decreased IgA/IgM and IgG/IgM ratios of antibodies specific to a variety of microbial and food antigens. The presented findings indicate reduced competence of mucosal B cells for class switch recombination from IgM to other isotypes limiting their capacity to react to changing antigenic variety in the gut lumen. Decreased availability of microbiota-specific IgA and IgG may be an important factor contributing to the translocation of microbial antigens across the intestinal mucosal barrier and their systemic dissemination that drives chronic inflammation in HIV-1-infected individuals. [ABSTRACT FROM AUTHOR]

Characterisation of the T cell receptors (TCR) involved in immune responses is important for the design of vaccines and immunotherapies for cancer and autoimmune disease. The specificity of the interaction between the TCR heterodimer and its peptide-MHC ligand derives largely from the juxtaposed hypervariable CDR3 regions on the TCRα and TCRβ chains, and obtaining the paired sequences of these regions is a standard for functionally defining the TCR. A brute force approach to identifying the TCRs in a population of T cells is to use high-throughput single-cell sequencing, but currently this process remains costly and risks missing small clones. Alternatively, CDR3α and CDR3β sequences can be associated using their frequency of co-occurrence in independent samples, but this approach can be confounded by the sharing of CDR3α and CDR3β across clones, commonly observed within epitope-specific T cell populations. The accurate, exhaustive, and economical recovery of TCR sequences from such populations therefore remains a challenging problem. Here we describe an algorithm for performing frequency-based pairing () that accommodates CDR3α- and CDR3β-sharing, cells expressing two TCRα chains, and multiple forms of sequencing error. The algorithm also yields accurate estimates of clonal frequencies. [ABSTRACT FROM AUTHOR]

Characterizing the immune response to pneumococcal proteins is critical in understanding this bacterium's epidemiology and vaccinology. Probing a custom-designed proteome microarray with sera from 35 healthy US adults revealed a continuous distribution of IgG affinities for 2,190 potential antigens from the species-wide pangenome. Reproducibly elevated IgG binding was elicited by 208 "antibody binding targets" (ABTs), which included 109 variants of the diverse pneumococcal surface proteins A and C (PspA and PspC) and zinc metalloprotease A and B (ZmpA and ZmpB) proteins. Functional analysis found ABTs were enriched inmotifs for secretion and cell surface association, with extensive representation of cell wall synthesis machinery, adhesins, transporter solute-binding proteins, and degradative enzymes. ABTs were associated with stronger evidence for evolving under positive selection, although this varied between functional categories, as did rates of diversification through recombination. Particularly rapid variation was observed at some immunogenic accessory loci, including a phage protein and a phase-variable glycosyltransferase ubiquitous among the diverse set of genomic islands encoding the serine-rich PsrP glycoprotein. Nevertheless, many antigens were conserved in the core genome, and strains' antigenic profiles were generally stable. No strong evidence was found for any epistasis between antigens driving population dynamics, or redundancy between functionally similar accessory ABTs, or age stratification of antigen profiles. These results highlight the paradox of why substantial variation is observed in only a subset of epitopes. This result may indicate only some interactions between immunoglobulins and ABTs clear pneumococcal colonization or that acquired immunity to pneumococci is an accumulation of individually weak responses to ABTs evolving under different levels of functional constraint. [ABSTRACT FROM AUTHOR]

Despite widespread uses of tetanus toxoid (TT) as a vaccine, model antigen and protein carrier, TT epitopes have been poorly characterized. Herein we defined the human CD4+ T cell epitope repertoire by reevaluation of previously described epitopes and evaluation of those derived from prediction of HLA Class II binding. Forty-seven epitopes were identified following in vitro TT stimulation, with 28 epitopes accounting for 90% of the total response. Despite this diverse range of epitopes, individual responses were associated with only a few immunodominant epitopes, with each donor responding on average to 3 epitopes. For the top 14 epitopes, HLA restriction could be inferred based on HLA typing of the responding donors. HLA binding predictions re-identified the vast majority of known epitopes, and identified 24 additional novel epitopes. With these epitopes, we created a TT epitope pool, which allowed us to characterize TT responses directly ex vivo using a cytokine-independent Activation Induced Marker (AIM) assay. These TT responses were highly Th1 or Th2 polarized, which was dependent upon the original priming vaccine, either the cellular DTwP or acellular DTaP formulation. This polarization remained despite the original priming having occurred decades past and a recent booster immunization with a reduced acellular vaccine formulation. While TT responses following booster vaccination were not durably increased in magnitude, they were associated with a relative expansion of CD4+ effector memory T cells. [ABSTRACT FROM AUTHOR]

Ruminants are the primary reservoir of Shiga-toxin producing Escherichia coli (STEC) O157:H7 and the main source of infection for humans. The aim of this study was to assess the immunogenic properties of a candidate vaccine consisting on the recombinant proteins of E. coli O157:H7 IntiminC280, the carboxy-terminal fraction of Intimin γ, EspB and the fusion protein between the B subunit of Stx2 and Brucella Lumazine Synthase (BLS)(BLS-Stx2B), in Holstein Fresian calves.To accomplish this goal we vaccinated calves with two doses of different vaccine formulations: 2 antigens (IntiminC280, EspB), 3 antigens (IntiminC280, EspB, BLS-Stx2B), BLS-Stx2B alone and a control non-vaccinated group. All antigens were expressed as recombinant proteins in E. coli. Specific IgG titres increased in vaccinated calves and the inclusion of BLS-Stx2B in the formulation seems to have a stimulatory effect on the humoral response to IntiminC280 and EspB after the booster. The neutralizing activity of antibodies against these two antigens was assessed in Red Blood Cell lysis assays and adherence to Hep-2 cells as a correlate of T3SS activity. Both sera from animals vaccinated with 2 or 3 antigens inhibited both virulence properties. Serological response to Stx2 was observed in animals vaccinated only with BLS-Stx2B and with 3 antigens and neutralization of Stx2 cytotoxicity was also observed in both groups. In conclusion, immunization of calves with BLS-Stx2B, IntiminC280 and EspB elicited a potent humoral response able to neutralize Shiga toxin 2 cytotoxity and the T3SS virulence properties in vitro. These results suggest that this formulation is a good candidate vaccine to reduce STEC shedding in cattle and needs to be further assessed in vivo. [ABSTRACT FROM AUTHOR]

Background: HIV-infected individuals have deficient responses to Yellow Fever vaccine (YFV) and may be at higher risk for adverse events (AE). Chronic immune activation–characterized by low CD4/CD8 ratio or high indoleamine 2,3-dioxygenase-1 (IDO) activity—may influence vaccine response in this population. Methods: We prospectively assessed AE, viremia by the YFV virus and YF-specific neutralizing antibodies (NAb) in HIV-infected (CD4>350) and -uninfected adults through 1 year after vaccination. The effect of HIV status on initial antibody response to YFV was measured during the first 3 months following vaccination, while the effect on persistence of antibody response was measured one year following vaccination. We explored CD4/CD8 ratio, IDO activity (plasma kynurenine/tryptophan [KT] ratio) and viremia by Human Pegivirus as potential predictors of NAb response to YFV among HIV-infected participants with linear mixed models. Results: 12 HIV-infected and 45-uninfected participants were included in the final analysis. HIV was not significantly associated with AE, YFV viremia or NAb titers through the first 3 months following vaccination. However, HIV–infected participants had 0.32 times the NAb titers observed for HIV-uninfected participants at 1 year following YFV (95% CI 0.13 to 0.83, p = 0.021), independent of sex, age and prior vaccination. In HIV-infected participants, each 10% increase in CD4/CD8 ratio predicted a mean 21% higher post-baseline YFV Nab titer (p = 0.024). Similarly, each 10% increase in KT ratio predicted a mean 21% lower post-baseline YFV Nab titer (p = 0.009). Viremia by Human Pegivirus was not significantly associated with NAb titers. Conclusions: HIV infection appears to decrease the durability of NAb responses to YFV, an effect that may be predicted by lower CD4/CD8 ratio or higher KT ratio. [ABSTRACT FROM AUTHOR]

Background. CD8+ T cells have putative roles in the regulation of adaptive immune responses during infection. The purpose of this paper is to compare the status of CD8+ T cells in Multiple Sclerosis (MS) and Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME). Methods. This preliminary investigation comprised 23 CFS/ME patients, 11 untreated MS patients, and 30 nonfatigued controls. Whole blood samples were collected from participants, stained with monoclonal antibodies, and analysed on the flow cytometer. Using the following CD markers, CD27 and CD45RA (CD45 exon isoform 4), CD8+ T cells were divided into naïve, central memory (CM), effector memory CD45RA- (EM), and effector memory CD45RA+ (EMRA) cells. Results. Surface expressions of BTLA, CD127, and CD49/CD29 were increased on subsets of CD8+ T cells from MS patients. In the CFS/ME patients CD127 was significantly decreased on all subsets of CD8+ T cells in comparison to the nonfatigued controls. PSGL-1 was significantly reduced in the CFS/ME patients in comparison to the nonfatigued controls. Conclusions. The results suggest significant deficits in the expression of receptors and adhesion molecules on subsets of CD8+ T cells in both MS and CFS/ME patients. These deficits reported may contribute to the pathogenesis of these diseases. However, larger sample size is warranted to confirm and support these encouraging preliminary findings. [ABSTRACT FROM AUTHOR]

We have investigated the immunogenicity in rabbits of native-like, soluble, recombinant SOSIP.664 trimers based on the env genes of four isolates of human immunodeficiency virus type 1 (HIV-1); specifically BG505 (clade A), B41 (clade B), CZA97 (clade C) and DU422 (clade C). The various trimers were delivered either simultaneously (as a mixture of clade A + B trimers) or sequentially over a 73-week period. Autologous, Tier-2 neutralizing antibody (NAb) responses were generated to the clade A and clade B trimers in the bivalent mixture. When delivered as boosting immunogens to rabbits immunized with the clade A and/or clade B trimers, the clade C trimers also generated autologous Tier-2 NAb responses, the CZA97 trimers doing so more strongly and consistently than the DU422 trimers. The clade C trimers also cross-boosted the pre-existing NAb responses to clade A and B trimers. We observed heterologous Tier-2 NAb responses albeit inconsistently, and with limited overall breath. However, cross-neutralization of the clade A BG505.T332N virus was consistently observed in rabbits immunized only with clade B trimers and then boosted with clade C trimers. The autologous NAbs induced by the BG505, B41 and CZA97 trimers predominantly recognized specific holes in the glycan shields of the cognate virus. The shared location of some of these holes may account for the observed cross-boosting effects and the heterologous neutralization of the BG505.T332N virus. These findings will guide the design of further experiments to determine whether and how multiple Env trimers can together induce more broadly neutralizing antibody responses. [ABSTRACT FROM AUTHOR]

Coxsackievirus A16 (CA16) is one of the major causative agents of hand, foot, and mouth disease worldwide. The non-neutralizing antibody response that targets CA16 VP1 remains poorly elucidated. In the present study, antibody responses against CA16 VP1 in Shanghai blood donors and Shanxi individuals were analyzed by ELISA and inhibitory ELISA using five CA16 VP1 antigens: VP11-297, VP141-297, VP11-60, VP145-58 and VP161-297. The correlation coefficients for most of the reactions against each of the five antigens and the inhibition of the anti-CA16 VP1 antibody response produced by the various antigens were higher in Shanghai blood donors compared to those in Shanxi individuals. VP11-297 and VP141-297 strongly inhibited the anti-CA16 VP1 response in serum samples from both populations, while VP145-58 and VP161-297 intermediately and weakly inhibited the anti-CA16 VP1 response, respectively, in only Shanghai group. A specific type of inhibition (anti-CA16 VP1 was completely inhibited by both VP11-60 and VP141-297) characterized by high neutralizing antibody titers was identified and accounted for 71.4% of the strongly reactive samples from the Shanghai group. These results indicate that the Shanghai blood donors exhibited a consistent and specific antibody response, while the Shanxi individuals showed an inconsistent and non-specific antibody response. These findings may improve the understanding of host humoral immunity against CA16 and help to identify an effective approach for seroepidemiological surveillance and specific diagnosis of CA16 infection based on normal and competitive ELISA. [ABSTRACT FROM AUTHOR]

Allogeneic stem cell transplantation can be a curative treatment for hematological malignancies. After HLA-matched allogeneic stem cell transplantation, beneficial anti-tumor immunity as well as detrimental side-effects can develop due to donor-derived T-cells recognizing polymorphic peptides that are presented by HLA on patient cells. Polymorphic peptides on patient cells that are recognized by specific T-cells are called minor histocompatibility antigens (MiHA), while the respective peptides in donor cells are allelic variants. MiHA can be identified by reverse strategies in which large sets of peptides are screened for T-cell recognition. In these strategies, selection of peptides by prediction algorithms may be relevant to increase the efficiency of MiHA discovery. We investigated the value of online prediction algorithms for MiHA discovery and determined the in silico characteristics of 68 autosomal HLA class I-restricted MiHA that have been identified as natural ligands by forward strategies in which T-cells from in vivo immune responses after allogeneic stem cell transplantation are used to identify the antigen. Our analysis showed that HLA class I binding was accurately predicted for 87% of MiHA of which a relatively large proportion of peptides had strong binding affinity (56%). Weak binding affinity was also predicted for a considerable number of antigens (31%) and the remaining 13% of MiHA were not predicted as HLA class I binding peptides. Besides prediction for HLA class I binding, none of the other online algorithms significantly contributed to MiHA characterization. Furthermore, we demonstrated that the majority of MiHA do not differ from their allelic variants in in silico characteristics, suggesting that allelic variants can potentially be processed and presented on the cell surface. In conclusion, our analyses revealed the in silico characteristics of 68 HLA class I-restricted MiHA and explored the value of online algorithms to predict T-cell ligands that are created by genetic variants. [ABSTRACT FROM AUTHOR]