DOCK2-deficiency causes defects in anti-viral T cell responses and impaired control of herpes simplex virus infection.

The expanding number of rare immunodeficiency syndromes offers an opportunity to understand key genes that support immune defence against infectious diseases. However, analysis of these in patients is complicated by their treatments and co-morbid infections requiring the use of mouse models for detailed investigations. Here we develop a mouse model of DOCK2 immunodeficiency and demonstrate that these mice have delayed clearance of herpes simplex virus type 1 (HSV-1) infections. We also uncovered a critical, cell intrinsic role of DOCK2 in the priming of anti-viral CD8+ T cells and in particular their initial expansion, despite apparently normal early activation of these cells. When this defect was overcome by priming in vitro, DOCK2-deficient CD8+ T cells were surprisingly protective against HSV-1-disease, albeit not as effectively as wild type cells. These results shed light on a cellular deficiency that is likely to impact anti-viral immunity in DOCK2-deficient patients.

DOCK2-deficiency causes defects in anti-viral T cell responses and poor control of herpes simplex virus infection.

The expanding number of rare immunodeficiency syndromes offers an opportunity to understand key genes that support immune defence against infectious diseases. However, patients with these diseases are by definition rare. In addition, any analysis is complicated by treatments and co-morbid infections requiring the use of mouse models for detailed investigations. Here we develop a mouse model of DOCK2 immunodeficiency and demonstrate that these mice have delayed clearance of herpes simplex virus type 1 (HSV-1) infections. Further, we found that they have a critical, cell intrinsic role of DOCK2 in the clonal expansion of anti-viral CD8+ T cells despite normal early activation of these cells. Finally, while the major deficiency is in clonal expansion, the ability of primed and expanded DOCK2-deficient CD8+ T cells to protect against HSV-1-infection is also compromised. These results provide a contributing cause for the frequent and devastating viral infections seen in DOCK2-deficient patients and improve our understanding of anti-viral CD8+ T cell immunity.

Surprisingly Effective Priming of CD8+ T Cells by Heat-Inactivated Vaccinia Virus Virions.

Robust priming of CD8+ T cells by viruses is considered to require infection and de novo expression of viral antigens. A corollary of this is that inactivated viruses are thought of as being inevitably poor vaccines for eliciting these responses. In contrast to this dogma, we found that some antigens present in vaccinia virus (VACV) virions prime strong CD8+ T cell responses when the virus was rendered noninfectious by heat. More surprisingly, in some cases these responses were similar in magnitude to those primed by infectious virus administered at an equivalent dose. Next, we tested whether this was a special property of particular antigens and their epitopes and found that foreign epitopes tagged onto three different VACV virion proteins were able to elicit CD8+ T cell responses irrespective of whether the virus was viable or heat killed. Further, the polyfunctionality and cytotoxic ability of the CD8+ T cells primed by these VACVs was equivalent irrespective of whether they were administered to mice as inactivated or live viruses. Finally, we used these VACVs in prime-boost combinations of inactivated and live virus and found that priming with dead virus before a live booster was the most immunogenic regime. We conclude that VACV virions can be efficient vectors for targeting antigens to dendritic cells for effective priming of CD8+ T cells, even when rendered noninfectious and speculate that this might also be the case for other viruses.IMPORTANCE The design of viral vectored vaccines is often considered to require a trade-off between efficacy and safety. This is especially the case for vaccines that aim to induce killer (CD8+) T cells, where there is a well-established dogma that links infection in vaccinated individuals with effective induction of immunity. However, we found that some proteins of vaccinia virus generate strong CD8+ T cell responses even when the virus preparation was inactivated by heat prior to administration as a vaccine. We took advantage of this finding by engineering a new vaccine vector virus that could be used as an inactivated vaccine. These results suggest that vaccinia virus may be a more versatile vaccine vector than previously appreciated and that in some instances safety can be prioritized by the complete elimination of viral replication without a proportional loss of immunogenicity.

Most viral peptides displayed by class I MHC on infected cells are immunogenic.

CD8+ T cells are essential effectors in antiviral immunity, recognizing short virus-derived peptides presented by MHC class I (pMHCI) on the surface of infected cells. However, the fraction of viral pMHCI on infected cells that are immunogenic has not been shown for any virus. To approach this fundamental question, we used peptide sequencing by high-resolution mass spectrometry to identify more than 170 vaccinia virus pMHCI presented on infected mouse cells. Next, we screened each peptide for immunogenicity in multiple virus-infected mice, revealing a wide range of immunogenicities. A surprisingly high fraction (>80%) of pMHCI were immunogenic in at least one infected mouse, and nearly 40% were immunogenic across more than half of the mice screened. The high number of peptides found to be immunogenic and the distribution of responses across mice give us insight into the specificity of antiviral CD8+ T cell responses.

Extent of Systemic Spread Determines CD8+ T Cell Immunodominance for Laboratory Strains, Smallpox Vaccines, and Zoonotic Isolates of Vaccinia Virus.

CD8(+) T cells that recognize virus-derived peptides presented on MHC class I are vital antiviral effectors. Such peptides presented by any given virus vary greatly in immunogenicity, allowing them to be ranked in an immunodominance hierarchy. However, the full range of parameters that determine immunodominance and the underlying mechanisms remain unknown. In this study, we show across a range of vaccinia virus strains, including the current clonal smallpox vaccine, that the ability of a strain to spread systemically correlated with reduced immunodominance. Reduction in immunodominance was observed both in the lymphoid system and at the primary site of infection. Mechanistically, reduced immunodominance was associated with more robust priming and especially priming in the spleen. Finally, we show this is not just a property of vaccine and laboratory strains of virus, because an association between virulence and immunodominance was also observed in isolates from an outbreak of zoonotic vaccinia virus that occurred in Brazil.

Delayed control of herpes simplex virus infection and impaired CD4(+) T-cell migration to the skin in mouse models of DOCK8 deficiency.

DOCK8 deficiency in humans and mice leads to multiple defects in immune cell numbers and function. Patients with this immunodeficiency have a high morbidity and mortality, and are distinguished by chronic cutaneous viral infections, including those caused by herpes simplex virus (HSV). The underlying mechanism of the specific susceptibility to these chronic cutaneous viral infections is currently unknown, largely because the effect of DOCK8 deficiency has not been studied in suitable models. A better understanding of these mechanisms is required to underpin the development of more specific therapies. Here we show that DOCK8-deficient mice have poor control of primary cutaneous herpes simplex lesions and this is associated with increased virus loads. Furthermore, DOCK8-deficient mice showed a lack of CD4(+) T-cell infiltration into HSV-infected skin.

Vaccinia virus F5 is required for normal plaque morphology in multiple cell lines but not replication in culture or virulence in mice.

Vaccinia virus (VACV) gene F5L was recently identified as a determinant of plaque morphology that is truncated in Modified Vaccinia virus Ankara (MVA). Here we show that F5L also affects plaque morphology of the virulent VACV strain Western Reserve (WR) in some, but not all cell lines, and not via previously described mechanisms. Further, despite a reduction in plaque size for VACV WR lacking F5L there was no evidence of reduced virus replication or spread in vitro or in vivo. In vivo we examined two mouse models, each with more than one dose and measured signs of disease and virus burden. These data provide an initial characterization of VACV F5L in a virulent strain of VACV. Further they show the necessity of testing plaque phenotypes in more than one cell type and provide an example of a VACV gene required for normal plaque morphology but not replication and spread.

An intact signal peptide on dengue virus E protein enhances immunogenicity for CD8(+) T cells and antibody when expressed from modified vaccinia Ankara.

Dengue is a global public health concern and this is aggravated by a lack of vaccines or antiviral therapies. Despite the well-known role of CD8(+) T cells in the immunopathogenesis of Dengue virus (DENV), only recent studies have highlighted the importance of this arm of the immune response in protection against the disease. Thus, the majority of DENV vaccine candidates are designed to achieve protective titers of neutralizing antibodies, with less regard for cellular responses. Here, we used a mouse model to investigate CD8(+) T cell and humoral responses to a set of potential DENV vaccines based on recombinant modified vaccinia virus Ankara (rMVA). To enable this study, we identified two CD8(+) T cell epitopes in the DENV-3 E protein in C57BL/6 mice. Using these we found that all the rMVA vaccines elicited DENV-specific CD8(+) T cells that were cytotoxic in vivo and polyfunctional in vitro. Moreover, vaccines expressing the E protein with an intact signal peptide sequence elicited more DENV-specific CD8(+) T cells than those expressing E proteins in the cytoplasm. Significantly, it was these same ER-targeted E protein vaccines that elicited antibody responses. Our results support the further development of rMVA vaccines expressing DENV E proteins and add to the tools available for dengue vaccine development.

Immunodomination during peripheral vaccinia virus infection.

Immunodominance is a fundamental property of CD8(+) T cell responses to viruses and vaccines. It had been observed that route of administration alters immunodominance after vaccinia virus (VACV) infection, but only a few epitopes were examined and no mechanism was provided. We re-visited this issue, examining a panel of 15 VACV epitopes and four routes, namely intradermal (i.d.), subcutaneous (s.c.), intraperitoneal (i.p.) and intravenous (i.v.) injection. We found that immunodominance is sharpened following peripheral routes of infection (i.d. and s.c.) compared with those that allow systemic virus dissemination (i.p. and i.v.). This increased immunodominance was demonstrated with native epitopes of VACV and with herpes simplex virus glycoprotein B when expressed from VACV. Responses to some subdominant epitopes were altered by as much as fourfold. Tracking of virus, examination of priming sites, and experiments restricting virus spread showed that priming of CD8(+) T cells in the spleen was necessary, but not sufficient to broaden responses. Further, we directly demonstrated that immunodomination occurs more readily when priming is mainly in lymph nodes. Finally, we were able to reduce immunodominance after i.d., but not i.p. infection, using a VACV expressing the costimulators CD80 (B7-1) and CD86 (B7-2), which is notable because VACV-based vaccines incorporating these molecules are in clinical trials. Taken together, our data indicate that resources for CD8(+) T cell priming are limiting in local draining lymph nodes, leading to greater immunodomination. Further, we provide evidence that costimulation can be a limiting factor that contributes to immunodomination. These results shed light on a possible mechanism of immunodomination and highlight the need to consider multiple epitopes across the spectrum of immunogenicities in studies aimed at understanding CD8(+) T cell immunity to viruses.

Kinetics of antigen expression and epitope presentation during virus infection.

Current knowledge about the dynamics of antigen presentation to T cells during viral infection is very poor despite being of fundamental importance to our understanding of anti-viral immunity. Here we use an advanced mass spectrometry method to simultaneously quantify the presentation of eight vaccinia virus peptide-MHC complexes (epitopes) on infected cells and the amounts of their source antigens at multiple times after infection. The results show a startling 1000-fold range in abundance as well as strikingly different kinetics across the epitopes monitored. The tight correlation between onset of protein expression and epitope display for most antigens provides the strongest support to date that antigen presentation is largely linked to translation and not later degradation of antigens. Finally, we show a complete disconnect between the epitope abundance and immunodominance hierarchy of these eight epitopes. This study highlights the complexity of viral antigen presentation by the host and demonstrates the weakness of simple models that assume total protein levels are directly linked to epitope presentation and immunogenicity.

Analyzing CD8 T cells in mouse models of poxvirus infection.

Mouse models of immunology are frequently used to study host responses to poxviruses or poxvirus-based recombinant vaccines. In this context, the magnitude of CD8(+) T cell responses is often of interest. Methods to evaluate CD8(+) T cell responses extend from those that rely on indirect measurement of effector function only, such as cytotoxicity assays, to those that only measure antiviral CD8(+) T cell numbers and not function, like peptide MHC tetramers. In this chapter, five methods are provided that cover this range: DimerX staining (a variant of peptide-MHC tetramers), intracellular cytokine staining for interferon-γ, CD62L/Granzyme B staining, and in vitro and ex vivo cytotoxicity assays. We also include tables of vaccinia virus peptide epitopes for use in most of these assays.

Linear fidelity in quantification of anti-viral CD8+ T cells.

Enumeration of anti-viral CD8(+) T cells to make comparisons between mice, viruses and vaccines is a frequently used approach, but controversy persists as to the most appropriate methods. Use of peptide-MHC tetramers (or variants) and intracellular staining for cytokines, in particular IFNγ, after a short ex vivo stimulation are now common, as are a variety of cytotoxicity assays, but few direct comparisons have been made. It has been argued that use of tetramers leads to the counting of non-functional T cells and that measurement of single cytokines will fail to identify cells with alternative functions. Further, the linear range of these methods has not been tested and this is required to give confidence that relative quantifications can be compared across samples. Here we show for two acute virus infections and CD8(+) T cells activated in vitro that DimerX (a tetramer variant) and intracellular staining for IFNγ, alone or in combination with CD107 to detect degranulation, gave comparable results at the peak of the response. Importantly, these methods were highly linear over nearly two orders of magnitude. In contrast, in vitro and in vivo assays for cytotoxicity were not linear, suffering from high background killing, plateaus in maximal killing and substantial underestimation of differences in magnitude of responses.

Analysis of A47, an immunoprevalent protein of vaccinia virus, leads to a reevaluation of the total antiviral CD8+ T cell response.

Vaccinia virus (VACV) is the prototypic orthopoxvirus and was the live vaccine used to eradicate smallpox. In addition, VACV is a possible vector for recombinant vaccines. Despite these reasons for study, the roles of many VACV genes are unknown, and some fundamental aspects, such as the total size of immune responses, remain poorly characterized. VACV gene A47L is of interest because it is highly transcribed, has no sequence similarity to any nonpoxvirus gene, and contains a larger-than-expected number of CD8(+) T cell epitopes. Here it is shown that A47L is not required for growth in vitro and does not contribute to virulence in mice. However, we confirmed that this one protein primes CD8(+) T cells to three different epitopes in C57BL/6 mice. In the process, one of these epitopes was redefined and shown to be the most dominant in A47 and one of the more highly ranked in VACV as a whole. The relatively high immunogenicity of this epitope led to a reevaluation of the total CD8(+) T cell response to VACV. By the use of two methods, the true size of the response was found to be around double previous estimates and at its peak is on the order of 60% of all CD8(+) T cells. We speculate that more CD8(+) T cell epitopes remain to be mapped for VACV and that underestimation of responses is unlikely to be unique to VACV, so there would be merit in revisiting this issue for other viruses.

Altered CD8(+) T cell immunodominance after vaccinia virus infection and the naive repertoire in inbred and F(1) mice.

Previous studies of CD8(+) T cell immunodominance after primary virus infection of F(1) mice compared with their inbred parents have generally concluded that no dramatic changes occur. In this study, we revisit this issue using vaccinia virus (VACV), which has a large genome, a recently defined immunodominance hierarchy in mice, and is a candidate vector for vaccines. We found that immunogenicity of VACV peptides defined using inbred mice was highly variable in F(1) progeny: some peptides were equally immunogenic in F(1) and inbred, whereas others elicited responses that were reduced by >90% in F(1) mice. Furthermore, the dominance of a peptide in the relevant inbred parent did not predict whether it would be poorly immunogenic in F(1) mice. This result held using F(1) hybrids of MHC-congenic mice, suggesting that MHC differences alone were responsible. It was also extended to foreign epitopes expressed by an rVACV vaccine. F(1) mice were less able to mount responses to the poorly immunogenic peptides when used as a sole immunogen, ruling out immunodomination. In addition, conserved TCR Vbeta usage between inbred and F(1) mice did not always correlate with strong responses in F(1) mice. However, direct estimation of naive precursor numbers showed that these were reduced in F(1) compared with inbred mice for specificities that were poorly immunogenic in the hybrids. These data have implications for our understanding of the extent to which MHC diversity alters the range of epitopes that are immunogenic in outbred populations.

Vaccinia virus CD8+ T-cell dominance hierarchies cannot be altered by prior immunization with individual peptides.

Heterologous prime-boost is a common vaccination strategy to elicit CD8(+) T cells (T(CD8+)), and vaccinia virus (VACV) has been widely used as a boosting vector. Studies with other viruses have suggested that priming may reduce responses to native epitopes in boosting vectors as well as improve responses to primed epitopes. We explored this possibility with a VACV model in mice and find that irrespective of an epitope's dominance, prior priming was able to double T(CD8+) responses. More surprisingly, and in contrast to findings for other viruses, responses to remaining epitopes were undisturbed, leaving the overall dominance hierarchy unchanged.

MIP-1alpha, MIP-1beta, RANTES, and ATAC/lymphotactin function together with IFN-gamma as type 1 cytokines.

We analyzed for the first time the expression of chemokines in subpopulations of the murine immune system at the single-cell level. We demonstrate in vitro and in a model of murine listeriosis that macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, regulated on activation normal T cell expressed and secreted (RANTES), and activation-induced, T cell-derived, and chemokine-related cytokine (ATAC)/lymphotactin are cosecreted to a high degree with IFN-gamma by activated individual natural killer (NK), CD8(+) T, and CD4(+) T helper 1 (Th1) cells. Functionally, ATAC and the CC chemokines cooperate with IFN-gamma in the up-regulation of CD40, IL-12, and tumor necrosis factor-alpha, molecules playing a central role in the effector phase of macrophages. Our data indicate that (i) MIP-1alpha, MIP-1beta, RANTES, and ATAC are not only chemoattractants but also coactivators of macrophages, (ii) MIP-1alpha, MIP-1beta, RANTES, and ATAC constitute together with IFN-gamma a group of "type 1 cytokines," and (iii) these cytokines act together as a functional unit that is used by NK cells in the innate phase and then "handed over" to CD8(+) T cells in the antigen-specific phase of the immune defense, thus bridging the two components of a Th1 immune reaction.