Modulating potency: Physicochemical characteristics are a determining factor of TLR4-agonist nanosuspension activity.

Activity of adjuvanted vaccines is difficult to predict in vitro and in vivo. The wide compositional and conformational range of formulated adjuvants, from aluminum salts to oil-in-water emulsions, makes comparisons between physicochemical and immunological properties difficult. Even within a formulated adjuvant class, excipient selection and concentration can alter potency and physicochemical properties of the mixture. Complete characterization of physicochemical properties of adjuvanted vaccine formulations and relationship to biological response is necessary to move beyond a guess-and-check paradigm toward directed development. Here we present a careful physicochemical characterization of a two-component nanosuspension containing synthetic TLR-4 agonist glucopyranosyl lipid adjuvant (GLA) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at various molar ratios. Physicochemical properties were compared with potency, as measured by stimulation of cytokine production in human whole blood. We found a surprising, nonlinear relationship between physicochemical properties and GLA-DPPC ratios that corresponded well with changes in biological activity. We discuss these data in light of the current understanding of TLR4 activation and the conformation-potency relationship in development of adjuvanted vaccines.

TLR4 ligand formulation causes distinct effects on antigen-specific cell-mediated and humoral immune responses.

The formulation of TLR ligands and other immunomodulators has a critical effect on their vaccine adjuvant activity. In this work, the synthetic TLR4 ligand GLA was formulated with three distinct vaccine delivery system platforms (aqueous suspension, liposome, or oil-in-water emulsion). The effect of the different formulations on the adaptive immune response to protein subunit vaccines was evaluated in the context of a recombinant malaria antigen, Plasmodium berghei circumsporozoite protein (PbCSP). Antibody responses in vaccinated mice were similar for the different formulations of GLA. However, cell-mediated responses differed significantly depending on the adjuvant system; in particular, the emulsion formulation of the TLR4 ligand induced significantly enhanced cellular IFN-γ and TNF-α responses compared to the other formulations. The effects of differences in adjuvant formulation composition and physical characteristics on biological activity are discussed. These results illustrate the importance of formulation of immunostimulatory adjuvants (e.g. TLR ligands) on the resulting immune responses to adjuvanted vaccines and may play a critical role for combating diseases where T cell immunity is advantageous.

Protection against tuberculosis with homologous or heterologous protein/vector vaccine approaches is not dependent on CD8+ T cells.

Considerable effort has been directed to develop Mycobacterium tuberculosis vaccines to boost bacille Calmette-Guérin or for those who cannot be immunized with bacille Calmette-Guérin. We hypothesized that CD4(+) and CD8(+) T cell responses with a heterologous prime/boost vaccine approach could induce long-lived vaccine efficacy against M. tuberculosis in C57BL/6 mice. We produced an adenovirus vector expressing ID93 (Ad5-ID93) for induction of CD8 T cells to use with our candidate tuberculosis vaccine, ID93/glucopyranosyl lipid adjuvant (GLA)-stable emulsion (SE), which induces potent Th1 CD4 T cells. Ad5-ID93 generates ID93-specific CD8(+) T cell responses and induces protection against M. tuberculosis. When Ad5-ID93 is administered in a prime-boost strategy with ID93/GLA-SE, both CD4(+) and CD8(+) T cells are generated and provide protection against M. tuberculosis. In a MHC class I-deficient mouse model, all groups including the Ad5-ID93 group elicited an Ag-specific CD4(+) T cell response and significantly fewer Ag-specific CD8(+) T cells, but were still protected against M. tuberculosis, suggesting that CD4(+) Th1 T cells could compensate for the loss of CD8(+) T cells. Lastly, the order of the heterologous immunizations was critical. Long-lived vaccine protection was observed only when Ad5-ID93 was given as the boost following an ID93/GLA-SE prime. The homologous ID93/GLA-SE prime/boost regimen also induced long-lived protection. One of the correlates of protection between these two approaches was an increase in the total number of ID93-specific IFN-γ-producing CD4(+) T cells 6 mo following the last immunization. Our findings provide insight into the development of vaccines not only for tuberculosis, but other diseases requiring T cell immunity.

Sculpting MHC class II-restricted self and non-self peptidome by the class I Ag-processing machinery and its impact on Th-cell responses.

It is generally assumed that the MHC class I antigen (Ag)-processing (CAP) machinery - which supplies peptides for presentation by class I molecules - plays no role in class II-restricted presentation of cytoplasmic Ags. In striking contrast to this assumption, we previously reported that proteasome inhibition, TAP deficiency or ERAAP deficiency led to dramatically altered T helper (Th)-cell responses to allograft (HY) and microbial (Listeria monocytogenes) Ags. Herein, we tested whether altered Ag processing and presentation, altered CD4(+) T-cell repertoire, or both underlay the above finding. We found that TAP deficiency and ERAAP deficiency dramatically altered the quality of class II-associated self peptides suggesting that the CAP machinery impacts class II-restricted Ag processing and presentation. Consistent with altered self peptidomes, the CD4(+) T-cell receptor repertoire of mice deficient in the CAP machinery substantially differed from that of WT animals resulting in altered CD4(+) T-cell Ag recognition patterns. These data suggest that TAP and ERAAP sculpt the class II-restricted peptidome, impacting the CD4(+) T-cell repertoire, and ultimately altering Th-cell responses. Together with our previous findings, these data suggest multiple CAP machinery components sequester or degrade MHC class II-restricted epitopes that would otherwise be capable of eliciting functional Th-cell responses.

Glucopyranosyl Lipid Adjuvant (GLA), a Synthetic TLR4 agonist, promotes potent systemic and mucosal responses to intranasal immunization with HIVgp140.

Successful vaccine development against HIV will likely require the induction of strong, long-lasting humoral and cellular immune responses in both the systemic and mucosal compartments. Based on the known immunological linkage between the upper-respiratory and urogenital tracts, we explored the potential of nasal adjuvants to boost immunization for the induction of vaginal and systemic immune responses to gp140. Mice were immunized intranasally with HIV gp140 together with micellar and emulsion formulations of a synthetic TLR4 agonist, Glucopyranosyl Lipid Adjuvant (GLA) and responses were compared to R848, a TLR7/8 agonist, or chitosan, a non TLR adjuvant. GLA and chitosan but not R848 greatly enhanced serum immunoglobulin levels when compared to antigen alone. Both GLA and chitosan induced high IgG and IgA titers in nasal and vaginal lavage and feces. The high IgA and IgG titers in vaginal lavage were associated with high numbers of gp140-specific antibody secreting cells in the genital tract. Whilst both GLA and chitosan induced T cell responses to immunization, GLA induced a stronger Th17 response and chitosan induced a more Th2 skewed response. Our results show that GLA is a highly potent intranasal adjuvant greatly enhancing humoral and cellular immune responses, both systemically and mucosally.

Intradermally administered TLR4 agonist GLA-SE enhances the capacity of human skin DCs to activate T cells and promotes emigration of Langerhans cells.

The natural TLR4 agonist lipopolysaccharide (LPS) has notable adjuvant activity. However, it is not useful as a vaccine adjuvant due to its toxicity. Glucopyranosyl lipid A (GLA) is a synthetic derivative of the lipid A tail of LPS with limited cytotoxicity, but strong potential to induce immune responses in mice, guinea pigs, non-human primates, and humans. In this study we determined how this synthetic TLR4 agonist affects the function of different subsets of human skin dendritic cells (DCs). The effect of GLA in an aqueous formulation (GLA-AF) or in an oil-in-water emulsion (GLA-SE) was compared to that of LPS and TLR3 agonist poly(I:C) using a human skin explant model with intradermal injections for the administration of the agonists. Intradermal injection of GLA-SE or LPS, but not GLA-AF, enhanced the emigration of CD1a(high)/langerin(+) Langerhans cells (LCs), but not dermal DCs (DDCs). LCs and CD14(-) DDCs exhibited an enhanced mature phenotype following intradermal administration of either of the two GLA formulations tested, similar to DCs that emigrated from LPS-injected skin. However, only injection of GLA-SE resulted in a significant increase in the production of the wide range of cytokines that is observed with LPS. Moreover, DCs that emigrated from GLA-SE-injected skin induced stronger CD4(+) T-cell activation, as indicated by a more pronounced T-cell proliferation, than DCs from skin injected with GLA-AF or LPS. Altogether, our data show that GLA-SE has a notable potency to stimulate the function of skin DCs, indicating that GLA-SE may be a good candidate as adjuvant for vaccines administered via the intradermal route.

Dissecting mechanisms of immunodominance to the common tuberculosis antigens ESAT-6, CFP10, Rv2031c (hspX), Rv2654c (TB7.7), and Rv1038c (EsxJ).

Diagnosis of tuberculosis often relies on the ex vivo IFN-γ release assays QuantiFERON-TB Gold In-Tube and T-SPOT.TB. However, understanding of the immunological mechanisms underlying their diagnostic use is still incomplete. Accordingly, we investigated T cell responses for the TB Ags included in the these assays and other commonly studied Ags: early secreted antigenic target 6 kDa, culture filtrate protein 10 kDa, Rv2031c, Rv2654c, and Rv1038c. PBMC from latently infected individuals were tested in ex vivo ELISPOT assays with overlapping peptides spanning the entirety of these Ags. We found striking variations in prevalence and magnitude of ex vivo reactivity, with culture filtrate protein 10 kDa being most dominant, followed by early secreted antigenic target 6 kDa and Rv2654c being virtually inactive. Rv2031c and Rv1038c were associated with intermediate patterns of reactivity. Further studies showed that low reactivity was not due to lack of HLA binding peptides, and high reactivity was associated with recognition of a few discrete dominant antigenic regions. Different donors recognized the same core sequence in a given epitope. In some cases, the identified epitopes were restricted by a single specific common HLA molecule (selective restriction), whereas in other cases, promiscuous restriction of the same epitope by multiple HLA molecules was apparent. Definition of the specific restricting HLA allowed to produce tetrameric reagents and showed that epitope-specific T cells recognizing either selectively or promiscuously restricted epitopes were predominantly T effector memory. In conclusion, these results highlight the feasibility of more clearly defined TB diagnostic reagent.

Targeting OX40 promotes lung-resident memory CD8 T cell populations that protect against respiratory poxvirus infection.

One goal of vaccination is to promote development of mucosal effector cells that can immediately respond to peripheral infection. This is especially important for protection against viruses that enter the host through the respiratory tract. We show that targeting the OX40 costimulatory receptor (CD134) strongly promotes mucosal memory in the CD8 T cell compartment. Systemic injection of an agonist antibody to OX40 strongly enhanced development of polyfunctional effector CD8 T cells that were induced after intraperitoneal infection with a highly virulent strain of vaccinia virus. These cells were located in lymphoid organs and also the lung, and importantly, long-term memory CD8 T cells were maintained in the lung over 1 year. Anti-OX40 also boosted memory development when mice were vaccinated subcutaneously with viral peptide. These CD8 T cells were sufficient to provide protection from lethal respiratory infection with live vaccinia virus independent of CD4 T cells and antibody. Again, the CD8 T cell populations that were induced after secondary infection displayed polyfunctionality and were maintained in the lung for over a year. These data suggest that agonists to the OX40 costimulatory receptor represent potential candidates for incorporation into vaccines for respiratory viruses.

Development and characterization of synthetic glucopyranosyl lipid adjuvant system as a vaccine adjuvant.

Innate immune responses to vaccine adjuvants based on lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls, are driven by Toll-like receptor (TLR) 4 and adaptor proteins including MyD88 and TRIF, leading to the production of inflammatory cytokines, type I interferons, and chemokines. We report here on the characterization of a synthetic hexaacylated lipid A derivative, denoted as glucopyranosyl lipid adjuvant (GLA). We assessed the effects of GLA on murine and human dendritic cells (DC) by combining microarray, mRNA and protein multiplex assays and flow cytometry analyses. We demonstrate that GLA has multifunctional immunomodulatory activity similar to naturally-derived monophosphory lipid A (MPL) on murine DC, including the production of inflammatory cytokines, chemokines, DC maturation and antigen-presenting functions. In contrast, hexaacylated GLA was overall more potent on a molar basis than heterogeneous MPL when tested on human DC and peripheral blood mononuclear cells (PBMC). When administered in vivo, GLA enhanced the immunogenicity of co-administered recombinant antigens, producing strong cell-mediated immunity and a qualitative T(H)1 response. We conclude that the GLA adjuvant stimulates and directs innate and adaptive immune responses by inducing DC maturation and the concomitant release of pro-inflammatory cytokines and chemokines associated with immune cell trafficking, activities which have important implications for the development of future vaccine adjuvants.

Uncovering the interplay between CD8, CD4 and antibody responses to complex pathogens.

Vaccinia virus (VACV) was used as the vaccine strain to eradicate smallpox. VACV is still administered to healthcare workers or researchers who are at risk of contracting the virus, and to military personnel. Thus, VACV represents a weapon against outbreaks, both natural (e.g., monkeypox) or man-made (bioterror). This virus is also used as a vector for experimental vaccine development (cancer/infectious disease). As a prototypic poxvirus, VACV is a model system for studying host-pathogen interactions. Until recently, little was known about the targets of host immune responses, which was likely owing to VACVs large genome (>200 open reading frames). However, the last few years have witnessed an explosion of data, and VACV has quickly become a useful model to study adaptive immune responses. This review summarizes and highlights key findings based on identification of VACV antigens targeted by the immune system (CD4, CD8 and antibodies) and the complex interplay between responses.

Definition of epitopes and antigens recognized by vaccinia specific immune responses: their conservation in variola virus sequences, and use as a model system to study complex pathogens.

In the last few years, a wealth of information has become available relating to the targets of vaccinia virus (VACV)-specific CD4(+) T cell, CD8(+) T cell and antibody responses. Due to the large size of its genome, encoding more than 200 different proteins, VACV represents a useful model system to study immunity to complex pathogens. Our data demonstrate that both cellular and humoral responses target a large number of antigens and epitopes. This broad spectrum of targets is detected in both mice and humans. CD4(+) T cell responses target late and structural antigens, while CD8(+) T cells preferentially recognize early antigens. While both CD4(+) and CD8(+) T cell responses target different types of antigens, the antigens recognized by T(H) cells are highly correlated with those recognized by antibody responses. We further show that protein abundance and antibody recognition can be used to predict antigens recognized by CD4(+) T cell responses, while early expression at the mRNA level predicts antigens targeted by CD8(+) T cells. Finally, we find that the vast majority of VACV epitopes are conserved in variola virus (VARV), thus suggesting that the epitopes defined herein also have relevance for the efficacy of VACV as a smallpox vaccine.

Correlates of protection efficacy induced by vaccinia virus-specific CD8+ T-cell epitopes in the murine intranasal challenge model.

The recent identification of a large array of different vaccinia virus-derived CD8(+) T-cell epitopes offers a unique opportunity to systematically analyze the correlation between protective efficacy and variables such as kinetics of expression and function of viral proteins, binding affinity to MHC molecules, immunogenicity, and viral antigen processing/presentation. In the current study, 49 different H-2(b) restricted epitopes were tested for their ability to protect peptide-immunized C57Bl/6 mice from lethal i.n. challenge with vaccinia virus. The epitopes varied greatly in their ability to confer protection, ranging from complete protection with minimal disease to no protection at all. The function or kinetics of the viral antigen expression did not correlate with protective efficacy. However, binding affinity partially predicted protection efficacy and ultimately epitope immunogenicity and recognition of infected cells offered the best correlation.

OX40 drives protective vaccinia virus-specific CD8 T cells.

Vaccinia virus (VACV) affords long-lasting protection against variola virus, the agent of smallpox. VACV-reactive CD8 T cells contribute to protection but their molecular control is unknown. We show that the TNFR molecule OX40 (CD134) controls primary VACV-specific CD8 T cell expansion and antiviral cytokine production and dictates development of strong memory to both dominant and subdominant VACV epitopes. Using adoptive transfer of OX40-deficient CD8 TCR-transgenic T cells responding to Ag in the context of VACV infection, we found that this reflects a direct action of OX40 expressed by CD8 T cells. Furthermore, CD8 T cells that can protect against lethal VACV challenge do not develop in mice deficient in OX40. Thus, OX40, which has been found to play little if any role in the generation of CD8 T cells to several viruses, including lymphocytic choriomeningitis virus and influenza, plays a dominant role in shaping the CD8 T cell response to VACV. These data suggest that unique costimulatory pathways might control alternate antiviral CD8 responses, demonstrating the plasticity of the immune response in utilizing different mechanisms to achieve similar ultimate goals.

Selective CD4+ T cell help for antibody responses to a large viral pathogen: deterministic linkage of specificities.

Antibody responses are critical components of protective immune responses to many pathogens, but parameters determining which proteins are targeted remain unclear. Vaccination with individual MHC-II-restricted vaccinia virus (VACV, smallpox vaccine) epitopes revealed that CD4(+) T cell help to B cells was surprisingly nontransferable to other virion protein specificities. Many VACV CD4(+) T cell responses identified in an unbiased screen targeted antibody virion protein targets, consistent with deterministic linkage between specificities. We tested the deterministic linkage model by efficiently predicting new vaccinia MHC II epitopes (830% improved efficiency). Finally, we showed CD4(+) T cell help was limiting for neutralizing antibody development and protective immunity in vivo. In contrast to the standard model, these data indicate individual proteins are the unit of B cell-T cell recognition for a large virus. Therefore, MHC restriction is a key selective event for the antiviral antibody response and is probably important for vaccine development to large pathogens.

Dissociation between epitope hierarchy and immunoprevalence in CD8 responses to vaccinia virus western reserve.

Understanding immunity to vaccinia virus (VACV) is important for the development of safer vaccines for smallpox- and poxvirus-vectored recombinant vaccines. VACV is also emerging as an outstanding model for studying CD8(+) T cell immunodominance because of the large number of CD8(+) T cell epitopes known for this virus in both mice and humans. In this study, we characterize the CD8(+) T cell response in vaccinated BALB/c mice by a genome-wide mapping approach. Responses to each of 54 newly identified H-2(d)-restricted T cell epitopes could be detected after i.p. and dermal vaccination routes. Analysis of these new epitopes in the context of those already known for VACV in mice and humans revealed two important findings. First, CD8(+) T cell epitopes are not randomly distributed across the VACV proteome, with some proteins being poorly or nonimmunogenic, while others are immunoprevalent, being frequently recognized across diverse MHC haplotypes. Second, some proteins constituted the major targets of the immune response by a specific haplotype as they recruited the majority of the specific CD8(+) T cells but these proteins did not correspond to the immunoprevalent Ags. Thus, we found a dissociation between immunoprevalence and immunodominance, implying that different sets of rules govern these two phenomena. Together, these findings have clear implications for the design of CD8(+) T cell subunit vaccines and in particular raise the exciting prospect of being able to choose subunits without reference to MHC restriction.

Vaccinia virus-specific CD4+ T cell responses target a set of antigens largely distinct from those targeted by CD8+ T cell responses.

Recent studies have defined vaccinia virus (VACV)-specific CD8(+) T cell epitopes in mice and humans. However, little is known about the epitope specificities of CD4(+) T cell responses. In this study, we identified 14 I-A(b)-restricted VACV-specific CD4(+) T cell epitopes by screening a large set of 2146 different 15-mer peptides in C57BL/6 mice. These epitopes account for approximately 20% of the total anti-VACV CD4(+) T cell response and are derived from 13 different viral proteins. Surprisingly, none of the CD4(+) T cell epitopes identified was derived from VACV virulence factors. Although early Ags were recognized, late Ags predominated as CD4(+) T cell targets. These results are in contrast to what was previously found in CD8(+) T cells responses, where early Ags, including virulence factors, were prominently recognized. Taken together, these results highlight fundamental differences in immunodominance of CD4(+) and CD8(+) T cell responses to a complex pathogen.

A consensus epitope prediction approach identifies the breadth of murine T(CD8+)-cell responses to vaccinia virus.

The value of predictive algorithms for identifying CD8+ T (T(CD8+))-cell epitopes has not been adequately tested experimentally. Here we demonstrate that conventional bioinformatic methods predict the vast majority of T(CD8+)-cell epitopes derived from vaccinia virus WR strain (VACV-WR) in the H-2(b) mouse model. This approach reveals the breadth of T-cell responses to vaccinia, a widely studied murine viral infection model, and may provide a tool for developing comprehensive antigenic maps of any complex pathogen.