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OBJECTIVE: To develop a passively targeted, patient-compliant, intranasal interleukin-10 (IL-10) gene therapy delivery system and to investigate its therapeutic benefit in experimental collagen-induced arthritis, a model of rheumatoid arthritis. METHODS: Arthritis was induced in DBA/1 mice and monitored following intranasal administration of an IL-10 plasmid (pG-IL-10) or the empty vector 2 days (days -2 and 19) prior to collagen injection (prophylactic group, as a single dose after collagen boost on day 21 (early therapy group, or as a single dose upon acquisition of a disease score of 3 (late therapy group. IL-10-induced alterations in cytokine secretion and proliferation by spleen and lymph node cells were assessed on days 31 and 65 and correlated with histologic changes and bone erosions assessed on day 65. RESULTS: Intranasal delivery of pG-IL-10 significantly delayed arthritis onset and reduced disease severity in the prophylactic group and early therapy group, reduced cellular infiltration and bone loss in the early therapy group, and reduced T cell proliferation in response to collagen on days 31 and 65 in these two groups, with a significant reduction in tumor necrosis factor alpha production on day 65. Within the late therapy group, disease progression was arrested for the rest of the study. The intranasally administered pG-IL-10 targeted monocytes and macrophages and showed dissemination to inflamed joints and draining lymph nodes in vivo. Importantly, systemic levels of IL-10 (in serum) were transient (peaking on day 2) and undetectable by day 4. CONCLUSION: Intranasal IL-10 gene delivery significantly reduces bone destruction, shows evidence of reducing joint inflammation, and may be mediated by high local levels of IL-10 produced by transfected monocytes trafficking to inflamed joints and draining lymph nodes. [ABSTRACT FROM AUTHOR]

This commentary provides an overview and links to presentations of a recent virtual congress series organized by the International Society for Vaccines (ISV) focused on COVID-19 vaccines. The series provided the academic community and vaccine developers as well as the wider general public with balanced information of the global response and resources for COVID-19 vaccines under development featuring: 1) NGOs and the regulatory perspective, 2) the status of vaccine development efforts, and 3) panel discussions to present and discuss challenges. ISV is a non-profit scientific organization whose members work on all areas relevant to vaccines. ISV plans to host additional virtual symposia including regional meetings and incorporating other topics along with COVID-19 vaccines.

A novel coronavirus, SARS-CoV-2, has recently emerged in China and spread internationally, posing a health emergency to the global community. COVID-19 caused by SARS-CoV-2 is associated with an acute respiratory illness that varies from mild to the life-threatening acute respiratory distress syndrome (ARDS). The complement system is part of the innate immune arsenal against pathogens, in which many viruses can evade or employ to mediate cell entry. The immunopathology and acute lung injury orchestrated through the influx of pro-inflammatory macrophages and neutrophils can be directly activated by complement components to prime an overzealous cytokine storm. The manifestations of severe COVID-19 such as the ARDS, sepsis and multiorgan failure have an established relationship with activation of the complement cascade. We have collected evidence from all the current studies we are aware of on SARS-CoV-2 immunopathogenesis and the preceding literature on SARS-CoV-1 and MERS-CoV infection linking severe COVID-19 disease directly with dysfunction of the complement pathways. This information lends support for a therapeutic anti-inflammatory strategy against complement, where a number of clinically ready potential therapeutic agents are available., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

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.

The generation of tissue resident memory (T RM ) 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., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: 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.

A simple dissolvable microneedle array (MA) platform has emerged as a promising technology for vaccine delivery, due to needle-free injection with a formulation that preserves the immunogenicity of live viral vectored vaccines dried in the MA matrix. While recent studies have focused largely on design parameters optimized to induce primary CD8(+) T cell responses, the hallmark of a vaccine is synonymous with engendering long-lasting memory. Here, we address the capacity of dried MA vaccination to programme phenotypic markers indicative of effector/memory CD8(+) T cell subsets and also responsiveness to recall antigen benchmarked against conventional intradermal (ID) injection. We show that despite a slightly lower frequency of dividing T cell receptor transgenic CD8(+) T cells in secondary lymphoid tissue at an early time point, the absolute number of CD8(+) T cells expressing an effector memory (CD62L(-)CD127(+)) and central memory (CD62L(+)CD127(+)) phenotype during peak expansion were comparable after MA and ID vaccination with a recombinant human adenovirus type 5 vector (AdHu5) encoding HIV-1 gag. Similarly, both vaccination routes generated CD8(+) memory T cell subsets detected in draining LNs for at least two years post-vaccination capable of responding to secondary antigen. These data suggest that CD8(+) T cell effector/memory generation and long-term memory is largely unaffected by physical differences in vaccine delivery to the skin via dried MA or ID suspension., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Despite the availability of major histocompatibility complex (MHC)-binding peptide prediction algorithms, the development of T-cell vaccines against pathogen and tumor antigens remains challenged by inefficient identification of immunogenic epitopes. CD8(+) T cells must distinguish immunogenic epitopes from nonimmunogenic self peptides to respond effectively against an antigen without endangering the viability of the host. Because this discrimination is fundamental to our understanding of immune recognition and critical for rational vaccine design, we interrogated the biochemical properties of 9,888 MHC class I peptides. We identified a strong bias toward hydrophobic amino acids at T-cell receptor contact residues within immunogenic epitopes of MHC allomorphs, which permitted us to develop and train a hydrophobicity-based artificial neural network (ANN-Hydro) to predict immunogenic epitopes. The immunogenicity model was validated in a blinded in vivo overlapping epitope discovery study of 364 peptides from three HIV-1 Gag protein variants. Applying the ANN-Hydro model on existing peptide-MHC algorithms consistently reduced the number of candidate peptides across multiple antigens and may provide a correlate with immunodominance. Hydrophobicity of TCR contact residues is a hallmark of immunogenic epitopes and marks a step toward eliminating the need for empirical epitope testing for vaccine development.

Summary Recombinant human binding immunoglobulin protein (BiP) has previously demonstrated anti-inflammatory properties in multiple models of inflammatory arthritis. We investigated whether these immunoregulatory properties could be exploited using gene therapy techniques. A single intraperitoneal injection of lentiviral vector containing the murine BiP (Lenti-mBiP) or green fluorescent protein (Lenti-GFP) transgene was administered in low- or high-dose studies during early arthritis. Disease activity was assessed by visual scoring, histology, serum cytokine and antibody production measured by cell enzyme-linked immunosorbent assay (ELISA) and ELISA, respectively. Lentiviral vector treatment caused significant induction of interferon (IFN)-γ responses regardless of the transgene; however, further specific effects were directly attributable to the BiP transgene. In both studies Lenti-mBiP suppressed clinical arthritis significantly. Histological examination showed that low-dose Lenti-mBiP suppressed inflammatory cell infiltration, cartilage destruction and significantly reduced pathogenic anti-type II collagen (CII) antibodies. Lenti-mBiP treatment caused significant up-regulation of soluble cytotoxic T lymphocyte antigen-4 (sCTLA-4) serum levels and down-regulation of interleukin (IL)-17A production in response to CII cell restimulation. In-vitro studies confirmed that Lenti-mBiP spleen cells could significantly suppress the release of IL-17A from CII primed responder cells following CII restimulation in vitro, and this suppression was associated with increased IL-10 production. Neutralization of CTLA-4 in further co-culture experiments demonstrated inverse regulation of IL-17A production. In conclusion, these data demonstrate proof of principle for the therapeutic potential of systemic lentiviral vector delivery of the BiP transgene leading to immunoregulation of arthritis by induction of soluble CTLA-4 and suppression of IL-17A production., (© 2014 British Society for Immunology.)

Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8(+) T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c(+) dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c(+) MHCII(hi) CD8α(neg) epithelial cell adhesion molecule (EpCAM(neg)) CD11b(+) langerin (Lang; CD207)(neg) DCs, but neither Langerhans cells nor Lang(+) DCs were required for CD8(+) T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8(+) T-cell priming by live rAdHu5 MAs.

There is an increasing body of evidence suggesting that the transfer of preformed MHC class I:peptide complexes between a virus-infected cell and an uninfected APC, termed cross-dressing, represents an important mechanism of Ag presentation to CD8+ T cells in host defense. However, although it has been shown that memory CD8+ T cells can be activated by uninfected dendritic cells (DCs) cross-dressed by Ag from virus-infected parenchymal cells, it is unknown whether conditions exist during virus infection in which naive CD8+ T cells are primed and differentiate to cytolytic effectors through cross-dressing, and indeed which DC subset would be responsible. In this study, we determine whether the transfer of MHC class I:peptide complexes between infected and uninfected murine DC plays a role in CD8+ T cell priming to viral Ags in vivo. We show that MHC class I:peptide complexes from peptide-pulsed or virus-infected DCs are indeed acquired by splenic CD8α⁻ DCs in vivo. Furthermore, the acquired MHC class I:peptide complexes are functional in that they induced Ag-specific CD8+ T cell effectors with cytolytic function. As CD8α⁻ DCs are poor cross-presenters, this may represent the main mechanism by which CD8α⁻ DCs present exogenously encountered Ag to CD8+ T cells. The sharing of Ag as preformed MHC class I:peptide complexes between infected and uninfected DCs without the restraints of Ag processing may have evolved to accurately amplify the response and also engage multiple DC subsets critical in the generation of strong antiviral immunity.

The title of this special topic invites us to identify areas in the field of IgA biology that are uncertain or in need of clarification. The inductive phase of the human intestinal IgA response has been a controversial area for some years. Therefore, to structure this review, we have identified key questions that are debated in this field. We have provided explanations of the origins of the uncertainties and have provided our own reasoned answers to the questions we pose.

It is uncertain whether NK cells modulate T cell memory differentiation. By using a genetic model that allows the selective depletion of NK cells, we show in this study that NK cells shape CD8(+) T cell fate by killing recently activated CD8(+) T cells in an NKG2D- and perforin-dependent manner. In the absence of NK cells, the differentiation of CD8(+) T cells is strongly biased toward a central memory T cell phenotype. Although, on a per-cell basis, memory CD8(+) T cells generated in the presence or the absence of NK cells have similar functional features and recall capabilities, NK cell deletion resulted in a significantly higher number of memory Ag-specific CD8(+) T cells, leading to more effective control of tumors carrying model Ags. The enhanced memory responses induced by the transient deletion of NK cells may provide a rational basis for the design of new vaccination strategies.

Dendritic cells (DCs) are crucial to intestinal immune regulation because of their roles in inducing protective immunity against pathogens while maintaining tolerance to commensal bacteria. Nonetheless, relatively little is known about intestinal DC responsiveness to innate immune stimuli via TLRs. We have previously shown that DCs migrating from the rat intestine in lymph (iLDCs) are hyporesponsive to LPS stimulation, thus possibly preventing harmful immune responses being induced to commensal flora. In this study, to understand how iLDC function is regulated by innate immune stimuli, we have characterized the expression and function of TLRs in iLDCs isolated from the thoracic duct lymph of mesenteric lymphadenectomized rats and compared these with DCs grown from bone marrow in the presence of Flt3 ligand. We show that iLDCs express mRNAs for all TLRs, but express significantly less TLR4 mRNA than bone marrow-derived DCs. Functionally, iLDCs could be activated by TLR agonists representing intestinal pathogen-associated molecular patterns, with the important exception of the TLR4 agonist LPS. Furthermore, we show that DCs in the intestinal wall interact directly with noninvasive bacteria (Bacillus subtilis spores), leading to an increase in the output of activated iLDCs into lymph, and that DCs containing spores are activated selectively. These data highlight a functional difference between TLR4 and other TLRs. As iLDCs can respond to TLR stimulation in vitro, there must be other mechanisms that prevent their activation by commensal bacteria under steady-state conditions.

There is a current need for safe, cheap, and effective vaccine adjuvants, to combine with sub-unit antigens to enhance their immunogenicity. In this study we have used probiotic Bacillus subtilis spores, known to be safe and fully tolerated by ingestion in man, and explored their ability to influence the magnitude and diversity of immune responses induced against two model antigens, tetanus toxoid fragment C (TT) and ovalbumin (OVA) in mice. The results show that B. subtilis spores not only increased antibody and T cell responses to a co-administered soluble antigen, but also broadened them, to include both antigen-specific CD4+ and CD8+ T cell responses as well as complement and non-complement fixing antibody isotypes. Furthermore, following intranasal immunization, spores augmented specific IgA to co-administered antigen both in the local respiratory and distal vaginal mucosa, as well as increased antigen-specific IgG antibody in draining LN and blood. Collectively, these data demonstrate that naturally occurring, non-pathogenic, non-commensal spores of B. subtilis both instruct and augment polyvalent immune responses and highlight their clinical potential in future vaccines to generate broad-based immunity.

Plasmacytoid dendritic cells (pDCs) recognize pathogen-associated molecules, particularly viral, and represent an important mechanism in innate defense. They may however, also have roles in steady-state tolerogenic responses at mucosal sites. pDCs can be isolated from blood, mucosa, and lymph nodes (LNs). Although pDCs can express peripherally derived Ags in LNs and at mucosal sites, it is not clear whether pDCs actually migrate from the periphery in lymph or whether LN pDCs acquire Ags by other mechanisms. To determine whether pDCs migrate in lymph, intestine or liver-draining LNs were removed and thoracic duct leukocytes (TDLs) were collected. TDLs expressing MHC-II and CD45R, but not TCRalphabeta or CD45RA, were then analyzed. These enriched TDLs neither transcribe type I IFNs nor secrete inflammatory cytokines in response to viral stimuli in vitro or after a TLR7/8 stimulus in vivo. In addition, these TDLs do not express CD5, CD90, CD200, or Siglec-H, but do express Ig, and therefore represent B cells, despite their lack of CD45RA expression. Intestinal and hepatic lymph are hence devoid of bona fide pDCs under both steady-state conditions and after TLR7/8 stimulation. This shows that any role for pDCs in Ag-specific T cell activation or tolerance must differ from the roles of classical dendritic cells, because it cannot result from peripheral Ag capture, followed by migration of pDCs via lymph to the LN.

The intestinal innate immune system continually interacts with commensal bacteria, thus oral vaccines should induce extra/alternative activation of DC, potentially through TLR. To examine this we collected intestinal lymph DC (iL-DC) under steady-state conditions and after feeding resiquimod (R-848), a synthetic TLR7/8 ligand, which we showed induces complete emptying of gut DC into lymph. iL-DC are heterogeneous with subset-specific functions. In this study we determined the kinetics of iL-DC subset release, activation and cytokine secretion induced by R-848. We show that L-DC comprise three distinct subsets (CD172ahigh, CD172aint and CD172alow) present with similar frequencies in intestinal but not hepatic lymph. No iL-DC express TLR7 mRNA, and only CD172a+ iL-DC express TLR8. However, after oral R-848 administration, output of all three subsets increases dramatically. CD172ahigh DC release precedes that of CD172alow DC, and the increased frequency of CD25high iL-DC is restricted to the two CD172a+ subsets. After feeding R-848 only CD172ahigh iL-DC secrete IL-6 and IL-12p40. However, CD172aint and CD172ahigh DC secrete similar but markedly lower amounts when stimulated in vitro. These results highlight the importance of in vivo approaches to assess adjuvant effects on DC and give novel insights into the subset-specific effects of an oral TLR ligand on intestinal DC.

Rheumatoid arthritis is a debilitating systemic autoimmune disease characterized by chronic synovial inflammation, which results in the progressive destruction of diseased joints. Advances in understanding the disease pathogenesis have led to the clinical introduction of biological inhibitors of inflammation or articular destruction. However, frequency of administration, cost and systemic side effects have driven efforts to develop gene therapeutic transfer strategies. This article reviews recent progress in the application of viral and non-viral vectors to target therapeutic genes for in vivo delivery.

DNA vaccines represent a new approach to the control of infectious disease. Both cellular and humoral immune responses are induced without the attendant concerns associated with live, attenuated vaccines. The vast majority of DNA vaccines are delivered by parental routes, which rarely elicit immune responses at the mucosal epithelia, the primary sites of pathogen transmission. In view of the importance of mucosal and regional lymph node immunity in the control of pathogens transmitted across the mucosal epithelia, a number of groups, including our own, have developed immunization strategies that target plasmid DNA to mucosal inductive sites associated with the lymphoid tissues of the respiratory, gastrointestinal, and genital tracts. Here, we describe the procedures for the formulation and delivery of plasmid DNA to mucosal inductive tissues and address the theoretical basis to selection of particular mucosal locations for the induction of effective immune responses.

The development of DNA vaccination to mucosal surfaces has continued apace over the last 2 years, with the investigation of several novel delivery vehicles. There have been advances in the understanding of the basic immunological mechanisms behind the induction of immune responses by plasmid DNA. The mechanistic insights are paving the way for the design of a second generation of mucosally delivered DNA vaccines. This article reviews the recent progress in the field of microparticle, cationic lipid and bacterial delivery systems. All these mechanisms afford some protection from environmental degradation and facilitate DNA uptake. These methods have been compared with respect to transfection efficiency, ability to elicit a full range of immune responses and their relative safety for in vivo applications.

The nasal mucosa provides a simple, non-invasive route to deliver DNA encoding the gene of interest to stimulate mucosal and systemic immune responses. However, unlike the intradermal or intramuscular routes for plasmid DNA (pDNA) delivery, immune responsiveness to antigen exposure at the respiratory mucosa is tightly regulated, consistent with the balance between active immunity and non-responsiveness to pathogenic or inert environmental antigens. We have characterised the antigen presenting cell types, their distribution and activation status following nasal vaccination with pDNA-cytofectin complexes encoding model antigens. We demonstrate that nasal immunisation is associated with expression of the encoded protein in a small population of dendritic cells and macrophages at the site of pDNA delivery, in the draining lymph nodes (LN) and in the spleen. Antigen expression by nasal dendritic cells was associated with up-regulation of surface MHC class II and CD86 expression and functional activation of T-lymphocytes. The results highlight the potential of intranasal vaccination with pDNA, provided the activation / costimulatory requirements for an active immune response are achieved.

The development of vaccines against pathogens transmitted across the genito-rectal mucosa that effectively stimulate both secretory IgA Abs and cytotoxic T lymphocytes in the genital tract and CTL in the draining lymph nodes (LN) has proven a major challenge. Here we report a novel, noninvasive approach of genetic vaccination via the intranasal route. Such vaccination elicits immune responses in the genital and rectal mucosa, draining LNs, and central lymphoid system. Intranasal immunization with plasmid DNA-lipid complexes encoding the model Ag firefly luciferase resulted in dissemination of the DNA and the encoded transcript throughout the respiratory and gastrointestinal tracts, draining LNs, and spleen. Complexing the plasmid DNA with the lipid DMRIE/DOPE enhanced expression of the encoded protein in the respiratory tract, increased specific secretory IgA Ab in the vaginal and rectal tracts, and increased the circulating levels of specific IgA and IgG. In addition, intranasal DNA immunization resulted in generation of Ag-specific CTL that were localized in the genital and cervical LNs and spleen. These results suggest that intranasal immunization with plasmid DNA-lipid complexes may represent a generic immunization strategy against pathogens transmitted across the genito-rectal and other mucosal surfaces.

The mucosal surfaces represent the primary site for transmission of several viruses including HIV. To prevent mucosal transmission and dissemination to the regional lymph nodes, an effective HIV vaccine may need to stimulate immune responses at the genital and rectal mucosa. Optimal induction of mucosal immunity in general requires targeting antigens to the specialized antigen presenting cells of mucosal associated lymphoid tissues. The nasal mucosa may provide a simple, non-invasive route to deliver DNA encoding the introduced gene to stimulate mucosal immunity. As a first step to evaluate the feasibility of this approach, we have investigated as a model system, systemic and mucosal immune responses elicited to firefly luciferase generated by DNA immunization. Incorporating DNA into liposomes with cationic lipids enhanced luciferase expression in nasal tissue, and was associated with induction of a humoral response in serum and vaginal fluids and also a proliferative and cytotoxic T lymphocyte response in the spleen and iliac lymph nodes draining the genital and rectal mucosa.

The major routes of HIV transmission are through the rectal and cervico-vaginal mucosa. To prevent dissemination of HIV to the regional lymph nodes (LNs), an effective vaccine may need to stimulate CTL in the rectal or genital tract and the draining LNs. We report that mucosal immunization by the recto-oral and vagino-oral route or s.c. immunization targeting the iliac LNs with a particulate SIVp27:Ty-VLP vaccine elicits SIVgag-specific CTL in the regional LNs as well as in the spleen and PBMC. Targeted LN immunization with this vaccine elicited MHC class I-restricted CD8+ CTL responses, and the highest frequency of CTL was found in the iliac LNs. Moreover, SIVgag-specific CTL activity was detected in short term T cell lines established in mononuclear cells eluted from the rectal and cervico-vaginal mucosa. The relative frequency of CTL in short term cell lines prepared from the rectal mucosa (21/113 or 18.6%) was similar to that obtained from the cervico-vaginal mucosa (16/79 or 20.3%). Examination of the relative frequency of CTL to the T cell epitopes residing within SIVp27 showed a higher frequency in iliac LN cells to peptide aa 41-70 than in that to peptide aa 121-150, and this was significant after both recto-oral (chi-squared 6.500, p < 0.02) and vagino-oral (chi-squared = 10.391, p < 0.01) immunization. In contrast, the relative frequency of CTL in PBMC to peptide aa 41-70 (15.5%) was comparable to that elicited by peptide aa 121-150 (17.6%). This study provides novel evidence that mucosal or targeted LN immunization can generate anti-SIV CTL in the rectal and genital mucosa, in the draining LNs, and in the central lymphoid system.

A s.c. route of immunization was developed in non-human primates, which targets the genitourinary-rectal associated lymphoid tissue. A vaccine consisting of rSIV gag p27, expressed as hybrid Ty virus-like particles (p27: Ty-VLP) was administered in the proximity of the internal iliac lymph nodes. Secretory IgA and IgG Abs to the p27 Ag were elicited in the vaginal, male urethral, rectal and seminal fluids, urine and serum. Two or more immunodominant B cell epitopes were identified within peptides 51-90 and 121-170 of the sequence of p27, using serum or biliary IgA and IgG Abs. CD4+ T cell proliferative responses to p27 were elicited predominantly in the targeted internal iliac, as well as the inferior mesenteric lymph nodes and the spleen, but not in the unrelated lymph nodes. These cells were then studied for helper function in p27 specific B cell Ab synthesis. Specific IgA and IgG Abs were detected in the same lymphoid tissues as those that displayed proliferative responses. However, cross-over reconstitution experiments between splenic and iliac lymph node B and CD4+ T cells suggest that the iliac B cells are essential for specific IgA Ab synthesis, whereas splenic B cells preferentially synthesize IgG Ab. The targeted lymph node (TLN) route of immunization gave comparable B cell, proliferative T cell, and Th cell responses to the vaginal, male genitourinary, and rectal mucosal routes, which were augmented by oral immunization. However, the TLN route induced urinary and seminal fluid sIgA and IgG Abs in addition to genital and rectal Abs. Generating secretory IgA and IgG Abs at the mucosal surfaces, and T and B cell immunity in the regional draining lymph nodes, spleen and circulation by TLN immunization may prevent transmission of virus through the mucosa, dissemination of the virus, and the formation of a latent reservoir of infection.

Human immunodeficiency virus (HIV) can be transmitted through infected seminal fluid or vaginal or rectal secretions during heterosexual or homosexual intercourse. To prevent mucosal transmission and spread to the regional lymph nodes, an effective vaccine may need to stimulate immune responses at the genitourinary mucosa. In this study, we have developed a mucosal model of genital immunization in male rhesus macaques, by topical urethral immunization with recombinant simian immunodeficiency virus p27gag, expressed as a hybrid Ty virus-like particle (Ty-VLP) and covalently linked to cholera toxin B subunit. This treatment was augmented by oral immunization with the same vaccine but with added killed cholera vibrios. Polymeric secretory immunoglobulin A (sIgA) and IgG antibodies to p27 were induced in urethral secretions, urine, and seminal fluid. This raises the possibility that the antibodies may function as a primary mucosal defense barrier against SIV (HIV) infection. The regional lymph nodes which constitute the genital-associated lymphoid tissue contained p27-specific CD4+ proliferative and helper T cells for antibody synthesis by B cells, which may function as a secondary immune barrier to infection. Blood and splenic lymphocytes also showed p27-sensitized CD4+ T cells and B cells in addition to serum IgG and IgA p27-specific antibodies; this constitutes a third level of immunity against dissemination of the virus. A comparison of genito-oral with recto-oral and intramuscular routes of immunization suggests that only genito-oral immunization elicits specific sIgA and IgG antibodies in the urine, urethra, and seminal fluid. Both genito-oral and recto-oral immunizations induced T-cell and B-cell immune responses in regional lymph nodes, with preferential IgA antibody synthesis. The mucosal route of immunization may prevent not only virus transmission through the genital mucosa but also dissemination and latency of the virus in the draining lymph nodes.

Transmission of human immunodeficiency virus (HIV) in North America and Europe occurs most commonly through the rectal mucosa during homosexual intercourse. The simian immunodeficiency virus (SIV) macaque model has been used to investigate rectal immunization. The vaccine used was a recombinant SIV gag p27 expressed as hybrid Ty virus-like particles (Ty-VLP). Sequential ororectal (OR) mucosal immunization was compared with i.m. immunization. Whereas both routes of immunization induced serum IgA and IgG p27 antibodies, only OR immunization induced rectal secretory IgA antibodies. Specific CD4+ T-cell proliferative responses to stimulation with p27 were found after i.m. immunization only in the blood and spleen, but after OR immunization they were found in the internal iliac and inferior mesenteric lymph nodes in addition to the blood and spleen. T-cell epitope mapping of the proliferative responses of short-term cell lines (STCLs) grown from peripheral blood or lymphoid cells revealed a major epitope within the polypeptide 121-150 after either route of immunization. Two minor T-cell epitopes were found within peptide 41-80 in STCLs from splenic and circulating cells. B-cell epitope mapping of serum or biliary IgA and IgG antibodies revealed two overlapping or adjacent immunodominant epitopes to the T-cell epitopes within the polypeptides 121-170 and 51-90. The results suggest that rectal augmented by oral immunization with a recombinant particulate antigen in nonhuman primates elicits secretory IgA and to a lesser extent IgG responses in the draining lymph nodes and the rectal mucosa, whereas systemic immunization targets predominantly splenic and circulating T- and B-cell responses. These findings may have important implications in the strategy of designing vaccines in prevention of homosexual transmission of HIV infection.

Heterosexual transmission through the cervico-vaginal mucosa is the principal route of human immunodeficiency virus (HIV) infection in Africa and is increasing in the United States and Europe. Vaginal immunization with simian immunodeficiency virus (SIV) had not yet been studied in nonhuman primates. Immune responses in macaques were investigated by stimulation of the genital and gut-associated lymphoid tissue with a recombinant, particulate SIV antigen. Vaginal, followed by oral, administration of the vaccine elicited three types of immunity: (i) gag protein p27-specific, secretory immunoglobulin A (IgA) and immunoglobulin G (IgG) in the vaginal fluid, (ii) specific CD4+ T cell proliferation and helper function in B cell p27-specific IgA synthesis in the genital lymph nodes, and (iii) specific serum IgA and IgG, with CD4+ T cell proliferative and helper functions in the circulating blood.

The UL13 open reading frame of herpes simplex virus type 1 (HSV-1) has been expressed in insect cells by a recombinant baculovirus and in Escherichia coli. In the latter case, the UL13 gene was fused to the gene for glutathione S-transferase (GST) to allow high-level expression of an 80-kDa GST-UL13 fusion protein. Antibody raised against the fusion protein reacted specifically with the 55-kDa UL13 gene product expressed by the recombinant baculovirus. This antibody also recognized a late phosphoprotein in HSV-1-infected cell lysates and a component of purified HSV-1 virions, both with the same electrophoretic mobility as the baculovirus-expressed protein. The virion component was efficiently phosphorylated in vitro by a virion-associated protein kinase. Using the same antibody, the probable homolog of the UL13 gene product was identified in HSV-2-infected cells and purified virions.

The outcome of infection by lymphocytic choriomeningitis virus (LCMV) is determined largely by the cytotoxic T lymphocyte (CTL) response of the host. In H-2b mice, the anti-glycoprotein (GP) response is directed to at least two epitopes, one located at GP aa 272-286 and a second in GP-1. Here we show that the second epitope can be minimally identified by amino acid residues GP 34-40 (AVYNFAT). The epitope is restricted by the Db class I glycoprotein. Characterization of these CTL epitopes allowed us to address the role(s) played by each epitope when expressed singly in the control of a lethal challenge with LCMV. Here we show that a single immunization with a recombinant vaccinia virus (VV) vaccine expressing LCMV GP aa 1-59 confers protection to H-2b mice from lethal LCMV infection. In contrast, a VV expressing LCMV GP aa 272-293, although recognized by CTL, does not protect. We show that the success or failure of protective immunization is determined by the ability of the immunizing sequences to prime for CTL in vivo. Although the GP 278-286 epitope when contained as a "minigene" fails to induce CTL, when incorporated in the normal GP "backbone" it successfully elicits CTL. These observations suggest that the "minimal" recognition sequence alone may not be sufficient to induce a protective CTL response in vivo. Thus a single CTL epitope can protect against a lethal virus infection, but to achieve an effective vaccine, the immunizing sequences must be carefully selected.

The possible role of viruses as the cause of some thyroid disorders was evaluated in three strains of mice neonatally infected with lymphocytic choriomeningitis virus. We report the first definitive evidence that viruses can persist in the thyroid gland, particularly thyroid epithelial cells in which thyroglobulin is synthesized. Concomitant with the infection of these cells was a significant reduction in the level of thyroglobulin mRNA and circulating thyroid hormones. Another virus that causes persistent infection but does not replicate in the thyroid gland failed to alter serum levels of thyroid hormones, indicating the thyroid dysfunction was not a generalized result of stress accompanying a persistent infection. This alteration in thyroid homeostasis during persistent infection with lymphocytic choriomeningitis virus is not caused by autoantibodies to the thyroid. Moreover, despite infection of the thyroid gland, neither necrosis nor inflammation occurs. Thyroid dysfunction was noted both when persistence was initiated at birth and in utero during congenital infection. These observations in an experimental model raise the issue that viruses may play a role in the pathogenesis of some thyroid disorders in man.

Serum antibodies to influenza A, measles, rubella, cytomegalovirus, varicella zoster, herpes simplex type 1, and mumps have been assayed in 104 patients with myasthenia gravis, grouped according to clinical features plus thymus pathology, and compared with matched controls. No significant differences in incidence or antibody titer were detected. In 37 patients with recent onset of symptoms, the incidence of antibody to coxsackieviruses B1-B6 was less than in controls. Juvenile-onset cases also demonstrated antibody to Epstein-Barr virus at the expected frequency. These results weaken the case for any of these common viruses, or the response to them, contributing to the pathogenesis of myasthenia gravis.

The efficiency of cloned class I MHC restricted CTL specific for the nucleoprotein or glycoprotein of lymphocytic choriomeningitis virus in either mediating virus clearance or immunopathologic disease in mice during acute infection was quantitated. Cloned CTL specific for either an internal (nucleoprotein) or surface (glycoprotein) protein of lymphocytic choriomeningitis virus, when administered intracerebrally 5 days after the initiation of infection induced fatal immunopathology, indicating that both internal and surface viral Ag play a role in immune mediated disease in vivo. Dose-response analysis indicated that only 10(2) to 10(3) cloned CTL injected intracerebrally were required to induce mortality in 50% of inoculated syngeneic mice. Thus relatively few virus-specific CTL are required to induce an acute immunopathologic disease in the central nervous system. In contrast, if cloned CTL are adoptively transferred at the time of initiation of viral infection they provide protection as demonstrated by their ability to eliminate virus from the brain and thus terminate the acute infection.

Virus-specific cytotoxic T lymphocytes (CTL) mediate their antiviral activity either by direct lysis of infected cells, or by the release of soluble lymphokines, or by a combination of the two. We have examined the role played by interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF alpha) in virus clearance. In vitro the amount of IFN-gamma synthesized by some lymphocytic choriomeningitis virus-specific H-2-restricted CTL clones was quantitatively too small to correlate with a direct antiviral activity in vivo. However, treatment of mice with a neutralizing monoclonal antibody to IFN-gamma significantly inhibited the clearance of virus from the spleens of acutely infected mice given adoptive transfers of immune spleen cells. Additionally, mice treated with exogenous recombinant murine IFN-gamma 24 h before or at the same time as virus inoculation showed reduced virus titres in their spleens. Hence, IFN-gamma displayed a direct antiviral effect in vivo. In contrast, treatment of mice with recombinant TNF alpha had no effect on virus clearance and thus TNF alpha is unlikely to play a significant role in this acute viral infection.

Persistent infection with lymphocytic choriomeningitis virus (LCMV) Armstrong strain in C3H/ST mice is associated with a growth hormone (GH) deficiency syndrome, characterized by growth retardation and low serum glucose levels (M.B.A. Oldstone et al., 1982, Science 218, 1125-1127). The syndrome is associated with a decrease in GH synthesis in the pituitary gland, in the absence of cellular injury in the pituitary or associated brain areas. In this report, we demonstrate that the decreases in steady-state synthesis of GH and its mRNA in virally infected mice is related to a 16-fold reduction in initiation of transcription of this gene. Minimal decrease, however, was demonstrated in transcriptional initiation of another pituitary gene, the precursor of thyroid simulating hormone (TSH-beta) or of the housekeeping genes, actin and pro alpha 2(I) collagen. Thus, viruses can cause disease in the absence of cytolytic or morphologic injury by selectively disrupting the synthesis of a differentiated cellular product at the level of transcriptional initiation.

Although viral infection has been suspected as the cause of some thyroid disorders, there has been limited data to support this contention seriously. Now we report the first evidence that lymphocytic choriomeningitis virus can persist in the thyroid gland, particularly thyroid epithelial cells in which thyroglobulin (Tg) the precursor of thyroid hormone, is synthesized. Concomitant with the infection of these cells is a significant reduction in Tg mRNA and in the level of circulating thyroid hormones. Another virus (lactate dehydrogenase virus) that causes persistent infection but does not replicate in the thyroid gland failed to alter levels of circulating thyroid hormones. These observations in an experimental model support the hypothesis that viruses may account for some thyroid disorders in man.

In a search for myotropic viruses with a potential to initiate muscle autoimmunity, we found that two strains of influenza A virus, A/England/863/78 (H3N2) and the reassortant virus X-47 (H3N2), could infect human syncytial myotubes lytically. The X-47 strain could, in addition, infect unicellular precursor myoblasts. Intracellular viral protein synthesis was demonstrated by pulse-labelling studies in both cell types with both virus strains. By immunofluorescence and immunoelectron microscopy, viral antigens were demonstrated in infected muscle cells specifically identified by double staining with monoclonal antibodies to either of two independent muscle-specific antigens. However, using 'co-capping' techniques in conjunction with electron microscopy, there was no evidence of association between viral haemagglutinin and the acetylcholine receptor (one major target of autoimmunity to muscle cells) on the infected cell membrane.

A systematic study of thymus homogenates and cell suspensions from 13 patients with myasthenia gravis (MG) of recent onset, and 6 non-myasthenic controls, has failed to detect or isolate virus by cell culture with 'rescue techniques', electron microscopy, or intracerebral inoculation into neonatal mice. These results do not support the case for persistent viral infection in the thymus, and impose constraints on hypotheses of a viral aetiology of MG.

CTL and NK cells cultured in vitro have been shown to contain a cytolytic pore-forming protein (PFP/perforin/cytolysin). To date, it has not been determined whether perforin is expressed by CTL that have been primed in vivo. Here, we have infected mice with two strains of lymphocytic choriomeningitis virus (LCMV), one of which mainly produces choriomeningitis and, the other, hepatitis. Brain and liver cryostat sections obtained from LCMV-infected mice were stained for various lymphocyte markers, including perforin. We were able to detect a large accumulation of perforin antigen in CD8+/Thy-1+/asialo GM1+/CD4- lymphocytes, which in fact represent the main infiltrating cell type found in brain and liver sections obtained during the late acute stage of LCMV infection. Perforin was also detected in a smaller population of CD8-/asialo GM1+/NK 1.1+/F480- cells, presumably corresponding to NK cells. Perforin-positive cells were found to have the morphology of blasts or large granular lymphocytes (LGL). These observations, together with in vitro studies performed in the past, indicate that perforin may be associated exclusively with LGL-like CTL blasts and NK cells. Our results demonstrate for the first time the presence of perforin in CTL that have been primed in vivo and suggest that perforin-positive CTL may be directly involved in producing the immunopathology associated with the LCMV infection.

Identification of a single viral T-cell epitope, associated with greater than 95% of the virus-specific cytotoxic T-lymphocyte (CTL) activity in BALB/c (H-2d) mice (J. L. Whitton, A. Tishon, H. Lewicki, J. Gebhard, T. Cook, M. Salvato, E. Joly, and M. B. A. Oldstone, J. Virol. 63:4303-4310, 1989), permitted us to design a CTL vaccine and test its ability to protect against a lethal virus challenge. Here we show that a single immunization with a recombinant vaccinia virus-lymphocytic choriomeningitis virus (LCMV) vaccine (VVNPaa1-201) expressing the immunodominant epitope completely protected H-2d mice from lethal infection with LCMV but did not protect H-2b mice. Furthermore, we show that the success or failure of immunization was determined entirely by the host class I major histocompatibility glycoproteins. The difference in outcome between mice of these two haplotypes was consistent with the presence or absence in the immunizing sequences of an epitope for CTL recognition and is correlated with the induction of LCMV-specific H-2-restricted CTL in H-2d mice. Protection is not conferred by a humoral immune response, since LCMV-specific antibodies were not detectable in sera from VVNPaa1-201-immunized mice. In addition, passive transfer of sera from vaccinated mice did not confer protection upon naive recipients challenged with LCMV. Hence, the molecular dissection of viral proteins can uncover immunodominant CTL epitope(s) that can be engineered into vaccines that elicit CTL. A single CTL epitope can protect against a lethal virus infection, but the efficacy of the vaccine varies in a major histocompatibility complex-dependent manner.