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Controlled human malaria infection (CHMI) provides a highly informative means to investigate host-pathogen interactions and enable in vivo proof-of-concept efficacy testing of new drugs and vaccines. However, unlike Plasmodium falciparum, well-characterized P. vivax parasites that are safe and suitable for use in modern CHMI models are limited. Here, two healthy malaria-naïve UK adults with universal donor blood group were safely infected with a clone of P. vivax from Thailand by mosquito-bite CHMI. Parasitemia developed in both volunteers and, prior to treatment, each volunteer donated blood to produce a cryopreserved stabilate of infected red blood cells. Following stringent safety screening, the parasite stabilate from one of these donors ("PvW1") was thawed and used to inoculate six healthy malaria-naïve UK adults by blood-stage CHMI, at three different dilutions. Parasitemia developed in all volunteers, who were then successfully drug treated. PvW1 parasite DNA was isolated and sequenced to produce a high quality genome assembly by using a hybrid assembly method. We analysed leading vaccine candidate antigens and multigene families, including the Vivax interspersed repeat (VIR) genes of which we identified 1145 in the PvW1 genome. Our genomic analysis will guide future assessment of candidate vaccines and drugs, as well as experimental medicine studies.

Plasmodium falciparum transmission-blocking vaccines (TBVs) targeting the Pfs25 antigen have shown promise in mice but the same efficacy has never been achieved in humans. We have previously published pre-clinical data related to a TBV candidate Pfs25-IMX313 encoded in viral vectors which was very promising and hence progressed to human clinical trials. The results from the clinical trial of this vaccine were very modest. Here we unravel why, contrary to mice, this vaccine has failed to induce robust antibody (Ab) titres in humans to elicit transmission-blocking activity. We examined Pfs25-specific B cell and T follicular helper (Tfh) cell responses in mice and humans after vaccination with Pfs25-IMX313 encoded by replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA) delivered in the heterologous prime-boost regimen via intramuscular route. We found that after vaccination, the Pfs25-IMX313 was immunologically suboptimal in humans compared to mice in terms of serum Ab production and antigen-specific B, CD4+ and Tfh cell responses. We identified that the key determinant for the poor anti-Pfs25 Ab formation in humans was the lack of CD4+ T cell recognition of Pfs25-IMX313 derived peptide epitopes. This is supported by correlations established between the ratio of proliferated antigen-specific CD4+/Tfh-like T cells, CXCL13 sera levels, and the corresponding numbers of circulating Pfs25-specific memory B cells, that consequently reflected on antigen-specific IgG sera levels. These correlations can inform the design of next-generation Pfs25-based vaccines for robust and durable blocking of malaria transmission.

Controlled human malaria infection (CHMI) provides a highly informative means to investigate host-pathogen interactions and enable in vivo proof-of-concept efficacy testing of new drugs and vaccines. However, unlike Plasmodium falciparum, well-characterized P. vivax parasites that are safe and suitable for use in modern CHMI models are limited. Here, two healthy malaria-naïve UK adults with universal donor blood group were safely infected with a clone of P. vivax from Thailand by mosquito-bite CHMI. Parasitemia developed in both volunteers and, prior to treatment, each volunteer donated blood to produce a cryopreserved stabilate of infected red blood cells. Following stringent safety screening, the parasite stabilate from one of these donors (“PvW1”) was thawed and used to inoculate six healthy malaria-naïve UK adults by blood-stage CHMI, at three different dilutions. Parasitemia developed in all volunteers, who were then successfully drug treated. PvW1 parasite DNA was isolated and sequenced to produce a high quality genome assembly by using a hybrid assembly method. We analysed leading vaccine candidate antigens and multigene families, including the Vivax interspersed repeat (VIR) genes of which we identified 1145 in the PvW1 genome. Our genomic analysis will guide future assessment of candidate vaccines and drugs, as well as experimental medicine studies.

BackgroundTransmission blocking vaccines targeting the sexual-stages of the malaria parasite could play a major role to achieve elimination and eradication of malaria. The Plasmodium falciparum Pfs25 protein (Pfs25) is the most clinically advanced candidate sexual-stage antigen. IMX313, a complement inhibitor C4b-binding protein that forms heptamers with the antigen fused to it, improve antibody responses. This is the first time that viral vectors have been used to induce antibodies in humans against an antigen that is expressed only in the mosquito vector.MethodsClinical trial looking at safety and immunogenicity of two recombinant viral vectored vaccines encoding Pfs25-IMX313 in healthy malaria-naive adults. Replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA), encoding Pfs25-IMX313, were delivered by the intramuscular route in a heterologous prime-boost regimen using an 8-week interval. Safety data and samples for immunogenicity assays were taken at various time-points.ResultsThe reactogenicity of the vaccines was similar to that seen in previous trials using the same viral vectors encoding other antigens. The vaccines were immunogenic and induced both antibody and T cell responses against Pfs25, but significant transmission reducing activity (TRA) was not observed in most volunteers by standard membrane feeding assay.ConclusionBoth vaccines were well tolerated and demonstrated a favorable safety profile in malaria-naive adults. However, the transmission reducing activity of the antibodies generated were weak, suggesting the need for an alternative vaccine formulation.Trial RegistrationClinicaltrials.gov NCT02532049.

Microneedle array platforms are a promising technology for vaccine delivery, due to their ease of administration with no sharp waste generated, small size, possibility of targeted delivery to the specified skin depth and efficacious delivery of different vaccine formulations, including viral vectors. Areas covered: Attributes and challenges of the most promising viral vector candidates that have advanced to the clinic and that have been leveraged for skin delivery by microneedles; The importance of understanding the immunobiology of antigen-presenting cells in the skin, in particular dendritic cells, in order to generate further improved skin vaccination strategies; recent studies where viral vectors expressing various antigens have been coupled with microneedle technology to examine their potential for improved vaccination. Expert opinion: Simple, economic and efficacious vaccine delivery methods are needed to improve health outcomes and manage possible outbreaks of new emerging viruses. Understanding what innate/inflammatory signals are required to induce both immediate and long-term responses remains a major hurdle in the development of the effective vaccines. One approach to meet these needs is microneedle-mediated viral vector vaccination. In order for this technology to fulfil this potential the industry must invest significantly to further develop its design, production, biosafety, delivery and large-scale manufacturing.

CD8+ T cells provide a critical defence from pathogens at mucosal epithelia including the female reproductive tract (FRT). Mucosal immunisation is considered essential to initiate this response, however this is difficult to reconcile with evidence that antigen delivered to skin can recruit protective CD8+ T cells to mucosal tissues. Here we dissect the underlying mechanism. We show that adenovirus serotype 5 (Ad5) bio-distributes at very low level to non-lymphoid tissues after skin immunisation. This drives the expansion and activation of CD3− NK1.1+ group 1 innate lymphoid cells (ILC1) within the FRT, essential for recruitment of CD8+ T-cell effectors. Interferon gamma produced by activated ILC1 is critical to licence CD11b+Ly6C+ monocyte production of CXCL9, a chemokine required to recruit skin primed CXCR3+ CD8+T-cells to the FRT. Our findings reveal a novel role for ILC1 to recruit effector CD8+ T-cells to prevent virus spread and establish immune surveillance at barrier tissues., Mucosal immunisation is important for initiating mucosal CD8+ T­cell responses but mucosal recruitment of protective CD8+ T cells can also be induced by skin immunisation. Here the authors examine the underlying mechanism and report a novel role for ILC1 recruiting CD8+ T cells to the mucosa after skin immunisation.

Dendritic cells (DCs) of the skin play an important role in skin-mediated immunity capable of promoting potent immune responses. We availed of polymeric dissolving microneedle (MN) arrays laden with nano-encapsulated antigen to specifically target skin DC networks. This modality of immunization represents an economic, efficient and potent means of antigen delivery directly to skin DCs, which are inefficiently targeted by more conventional immunization routes. Following MN immunization, Langerhans cells (LCs) constituted the major skin DC subset capable of cross-priming antigen-specific CD8(+) T cells ex-vivo. While all DC subsets were equally efficient in priming CD4(+) T cells, LCs were largely responsible for orchestrating the differentiation of CD4(+) IFN-γ and IL-17 producing effectors. Importantly, depletion of LCs prior to immunization had a profound effect on CD8(+) CTL responses in vivo, and vaccinated animals displayed reduced protective anti-tumour and viral immunity. Interestingly, this cross-priming bias was lost following MN immunization with soluble antigen, suggesting that processing and cross-presentation of nano-particulate antigen is favoured by LCs. Therefore, these studies highlight the importance of LCs in skin immunization strategies and that targeting of nano-particulate immunogens through dissolvable polymeric MNs potentially provides a promising technological platform for improved vaccination strategies.Journal of Investigative Dermatology accepted article preview online, 22 September 2014. doi:10.1038/jid.2014.415.

The generation of tissue resident memory (TRM) cells at the body surfaces to provide a front line defence against invading pathogens represents an important goal in vaccine development for a wide variety of pathogens. It has been widely assumed that local vaccine delivery to the mucosae is necessary to achieve that aim. Here we characterise a novel micro-needle array (MA) delivery system fabricated to deliver a live recombinant human adenovirus type 5 vaccine vector (AdHu5) encoding HIV-1 gag. We demonstrate rapid dissolution kinetics of the microneedles in skin. Moreover, a consequence of MA vaccine cargo release was the generation of long-lived antigen-specific CD8+ T cells that accumulate in mucosal tissues, including the female genital and respiratory tract. The memory CD8+ T cell population maintained in the peripheral mucosal tissues was attributable to a MA delivered AdHu5 vaccine instructing CD8+ T cell expression of CXCR3+, CD103+, CD49a+, CD69+, CD127+ homing, retention and survival markers. Furthermore, memory CD8+ T cells generated by MA immunization significantly expanded upon locally administered antigenic challenge and showed a predominant poly-functional profile producing high levels of IFNγ and Granzyme B. These data demonstrate that skin vaccine delivery using microneedle technology induces mobilization of long lived, poly-functional CD8+ T cells to peripheral tissues, phenotypically displaying hallmarks of residency and yields new insights into how to design and deliver effective vaccine candidates with properties to exert local immunosurveillance at the mucosal surfaces., Graphical abstract Image 1

The efficacious delivery of antigens to antigen-presenting cells (APCs), in particular, to dendritic cells (DCs), and their subsequent activation remains a significant challenge in the development of effective vaccines. This study highlights the potential of dissolving microneedle (MN) arrays laden with nanoencapsulated antigen to increase vaccine immunogenicity by targeting antigen specifically to contiguous DC networks within the skin. Following in situ uptake, skin-resident DCs were able to deliver antigen-encapsulated poly-d,l-lactide-co-glycolide (PGLA) nanoparticles to cutaneous draining lymph nodes where they subsequently induced significant expansion of antigen-specific T cells. Moreover, we show that antigen-encapsulated nanoparticle vaccination via microneedles generated robust antigen-specific cellular immune responses in mice. This approach provided complete protection in vivo against both the development of antigen-expressing B16 melanoma tumors and a murine model of para-influenza, through the activation of antigen-specific cytotoxic CD8(+) T cells that resulted in efficient clearance of tumors and virus, respectively. In addition, we show promising findings that nanoencapsulation facilitates antigen retention into skin layers and provides antigen stability in microneedles. Therefore, the use of biodegradable polymeric nanoparticles for selective targeting of antigen to skin DC subsets through dissolvable MNs provides a promising technology for improved vaccination efficacy, compliance, and coverage.

OBJECTIVES The aims of this study were to estimate the level of knowledge, attitudes, and intentions about human papillomavirus (HPV) infection and HPV vaccine among gynecologists and to explore predictors of gynecologists' intention to recommend the HPV vaccine. METHOD This research was conducted between April and June 2014 among all gynecologists working at women's health departments in all primary health centers in Belgrade, the capital of Serbia, using a specially designed questionnaire. RESULTS The response rate was 88.2%. The knowledge of gynecologists was estimated as average. The most frequently reported obstacles to HPV vaccination was the financial concern (59.8%). More than two thirds of the gynecologists were willing to recommend the vaccine (68.4%). The factors associated with the gynecologists' intention to recommend the vaccine included their positive attitudes toward boys' vaccination (odds ratio [OR], 8.96; 95% confidence interval [CI], 2.85-28.16), negative attitudes toward frequent changes the recommendations (OR, 0.31; 95% CI, 0.10-0.93), and beliefs that the vaccine application would decrease condom usage (OR, 0.31; 95% CI, 0.06-0.68). CONCLUSIONS The findings provide an important insight into the current point of view of the gynecologists, which confirms that the better the knowledge of HPV and vaccine, the higher the likelihood of recommending it.

Induction of endotoxin tolerance leads to a reduced inflammatory response after repeated challenge by LPS and is important for resolution of inflammation and prevention of tissue damage. Enterobacterial LPS is recognized by the TLR4 signaling complex, whereas LPS of some non-enterobacterial organisms is capable of signaling independently of TLR4 utilizing TLR2-mediated signal transduction instead. In this study we report that Porphyromonas gingivalis LPS, a TLR2 agonist, fails to induce a fully endotoxin tolerant state in a human monocytic cell line (THP-1) and mouse bone marrow-derived macrophages. In contrast to significantly decreased production of human IL-8 and TNF-α and, in mice, keratinocyte-derived cytokine (KC), macrophage inflammatory protein-2 (MIP-2), and TNF-α after repeated challenge with Escherichia coli LPS, cells repeatedly exposed to P. gingivalis LPS responded by producing less TNF-α but sustained elevated secretion of IL-8, KC, and MIP-2. Furthermore, in endotoxin-tolerant cells, production of IL-8 is controlled at the signaling level and correlates well with NF-κB activation, whereas TNF-α expression is blocked at the gene transcription level. Interferon β plays an important role in attenuation of chemokine expression in endotoxin-tolerized cells as shown in interferon regulatory factor-3 knock-out mice. In addition, human gingival fibroblasts, commonly known not to display LPS tolerance, were found to be tolerant to repeated challenge by LPS if pretreated with interferon β. The data suggest that the inability of the LPS-TLR2 complex to induce full endotoxin tolerance in monocytes/macrophages is related to diminished production of interferon β and may partly explain the involvement of these LPS isoforms in the pathogenesis of chronic inflammatory diseases.

Objectives We aimed to highlight the utility of novel dissolving microneedle (MN)-based delivery systems for enhanced transdermal protein delivery. Vaccination remains the most accepted and effective approach in offering protection from infectious diseases. In recent years, much interest has focused on the possibility of using minimally invasive MN technologies to replace conventional hypodermic vaccine injections. Methods The focus of this study was exploitation of dissolving MN array devices fabricated from 20% w/w poly(methyl vinyl ether/maleic acid) using a micromoulding technique, for the facilitated delivery of a model antigen, ovalbumin (OVA). Key findings A series of in-vitro and in-vivo experiments were designed to demonstrate that MN arrays loaded with OVA penetrated the stratum corneum and delivered their payload systemically. The latter was evidenced by the activation of both humoral and cellular inflammatory responses in mice, indicated by the production of immunoglobulins (IgG, IgG1, IgG2a) and inflammatory cytokines, specifically interferon-gamma and interleukin-4. Importantly, the structural integrity of the OVA following incorporation into the MN arrays was maintained. Conclusion While enhanced manufacturing strategies are required to improve delivery efficiency and reduce waste, dissolving MN are a promising candidate for ‘reduced-risk’ vaccination and protein delivery strategies.

Breaching the skin’s stratum corneum barrier raises the possibility of administration of vaccines, gene vectors, antibodies, photosensitisers and even nanoparticles, all of which have at least their initial effect on populations of skin cells. Intradermal vaccine delivery, in particular, holds enormous potential for improved therapeutic outcomes for patients, particularly those in the developing world. Various microneedle-based vaccine delivery strategies have been employed, and here we discuss each one in turn. We also describe the importance of cutaneous immunobiology on the effect produced by microneedle-mediated intradermal vaccination.

Breaching the skin's stratum corneum barrier raises the possibility of the administration of vaccines, gene vectors, antibodies and even nanoparticles, all of which have at least their initial effect on populations of skin cells.Intradermal vaccine delivery holds enormous potential for improved therapeutic outcomes for patients, particularly those in the developing world. Various vaccine-delivery strategies have been employed, which are discussed in this review. The importance of cutaneous immunobiology on the effect produced by microneedle-mediated intradermal vaccination is also discussed.Microneedle-mediated vaccines hold enormous potential for patient benefit. However, in order for microneedle vaccine strategies to fulfill their potential, the proportion of an immune response that is due to the local action of delivered vaccines on skin antigen-presenting cells, and what is due to a systemic effect from vaccines reaching the systemic circulation, must be determined. Moreover, industry will need to invest significantly in new equipment and instrumentation in order to mass-produce microneedle vaccines consistently. Finally, microneedles will need to demonstrate consistent dose delivery across patient groups and match this to reliable immune responses before they will replace tried-and-tested needle-and-syringe-based approaches.