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Available COVID-19 vaccine only provide protection for a limited time due in part to the rapid emergence of viral variants with spike protein mutations, necessitating the generation of new vaccines to combat SARS-CoV-2. Two serologically distinct replication-defective chimpanzee-origin adenovirus (Ad) vectors (AdC) called AdC6 and AdC7 expressing early SARS-CoV-2 isolate spike (S) or nucleocapsid (N) proteins, the latter expressed as a fusion protein within herpes simplex virus glycoprotein D (gD), were tested individually or as a mixture in a hamster COVID-19 SARS-CoV-2 challenge model. The S protein expressing AdC (AdC-S) vectors induced antibodies including those with neutralizing activity that in part cross-reacted with viral variants. Hamsters vaccinated with the AdC-S vectors were protected against serious disease and showed accelerated recovery upon SARS-CoV-2 challenge. Protection was enhanced if AdC-S vectors were given together with the AdC vaccines that expressed the gD N fusion protein (AdC-gDN). In contrast hamsters that just received the AdC-gDN vaccines showed only marginal lessening of symptoms compared to control animals. These results indicate that immune response to the N protein that is less variable than the S protein may potentiate and prolong protection achieved by the currently used S protein based genetic COVID-19 vaccines.

Two serologically distinct replication-defective chimpanzee-origin adenovirus (Ad) vectors (AdC) called AdC6 and AdC7 expressing the spike (S) or nucleocapsid (N) proteins of an early SARS-CoV-2 isolate were tested individually or as a mixture in a hamster COVID-19 challenge model. The N protein, which was expressed as a fusion protein within herpes simplex virus glycoprotein D (gD) stimulated antibodies and CD8+ T cells. The S protein expressing AdC (AdC-S) vectors induced antibodies including those with neutralizing activity that in part cross-reacted with viral variants. Hamsters vaccinated with the AdC-S vectors were protected against serious disease and showed accelerated recovery upon SARS-CoV-2 challenge. Protection was enhanced if AdC-S vectors were given together with the AdC vaccines that expressed the gDN fusion protein (AdC-gDN). In contrast hamsters that just received the AdC-gDN vaccines showed only marginal lessening of symptoms compared to control animals. These results indicate that immune response to the N protein that is less variable that the S protein may potentiate and prolong protection achieved by the currently used genetic COVID-19 vaccines.

Two serologically distinct chimpanzee-origin, replication-defective adenovirus (AdC) vectors expressing the spike (S) protein of an early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate were generated and tested for induction of antibodies in young and aged mice. Both vectors induced S protein-specific antibodies including neutralizing antibodies. Levels of antibodies increased after a boost. The effectiveness of the boost depended on vector dose and timing between the two immunizations. Using two heterologous AdC vectors was more effective than vaccinating with the same vector repeatedly. Antibodies partially crossreacted between different S protein variants. Cross-reactivity increased after booster immunization with vectors carrying the same S gene, expression of two different S proteins by the AdC vectors used for the prime and the boost did not selectively increase responses against the variants.

Adenovirus (Ad) is used extensively for construction of viral vectors, most commonly with deletion in its E1 and/or E3 genomic regions. Previously, our attempts to insert envelope proteins (Env) of HIV-1 into such vectors based on chimpanzee-derived Ad (AdC) viruses were thwarted. Here, we describe that genetic instability of an E1- and E3-deleted AdC vector of serotype C6 expressing Env of HIV-1 can be overcome by reinsertion of E3 sequences with anti-apoptotic activities. This partial E3 deletion presumably delays premature death of HEK-293 packaging cell lines due to Env-induced cell apoptosis. The same partial E3 deletion also allows for the generation of stable glycoprotein 140 (gp140)- and gp160-expressing Ad vectors based on AdC7, a distinct AdC serotype. Env-expressing AdC vectors containing the partial E3 deletion are genetically stable upon serial cell culture passaging, produce yields comparable to those of other AdC vectors, and induce transgene product-specific antibody responses in mice. A partial E3 deletion thereby allows expansion of the repertoire of transgenes that can be expressed by Ad vectors.

To determine if an ordered and repetitive display of an epitope promoted induction of superior antibody responses, we compared B-cell responses to an influenza A virus epitope that was either encoded as a transgene by an adenovirus (Ad) vector or expressed on the vector's surface. To this end, we constructed a panel of influenza A virus vaccines based on chimpanzee-derived replication-defective adenovirus (AdC) vectors of serotype SAd-V25 also called AdC68. AdC68 vectors were modified to express a linear B-cell epitope of the ectodomain of matrix 2 (M2e) within variable regions 1 (VR1) or 4 (VR4) of the adenovirus hexon. Additional vectors with wild-type or M2e-modified hexon encoded M2e fused to the influenza A virus nucleoprotein (NP) as a transgene product. Hexon-modified vectors were tested for immunogenicity and efficacy in mice in comparison to vectors with native hexon expressing the M2e-NP fusion protein. Upon priming, vectors expressing M2e within VR1 of hexon induced M2e-specific antibody responses of higher magnitude and avidity than those carrying M2e within VR4 or vectors expressing the M2e as part of a transgene product. CD8(+) T-cell responses to the transgenic NP were comparable between vectors. M2e-specific antibody responses could be boosted by a second dose of the VR1 hexon-modified vector but not by repeated immunization with the VR4 hexon-modified vector.

How tumor-infiltrating T lymphocytes (TILs) adapt to the metabolic constrains within the tumor microenvironment (TME) and to what degree this affects their ability to combat tumor progression remain poorly understood. Using mouse melanoma models, we report that CD8

In animal models, E1-deleted human adenoviral recombinants of the serotype 5 (AdHu5) have shown high efficacy as vaccine carriers for different Ags including those of HIV-1. Humans are infected by common serotypes of human adenovirus such as AdHu5 early in life and a significant percentage has high levels of neutralizing Abs to these serotypes, which will very likely impair the efficacy of recombinant vaccines based on the homologous virus. To circumvent this problem, a novel replication-defective adenoviral vaccine carrier based on an E1-deleted recombinant of the chimpanzee adenovirus 68 (AdC68) was developed. An AdC68 construct expressing a codon-optimized, truncated form of gag of HIV-1 induces CD8+ T cells to gag in mice which at the height of the immune response encompass nearly 20% of the entire splenic CD8+ T cell population. The vaccine-induced immune response provides protection to challenge with a vaccinia gag recombinant virus. Induction of transgene-specific CD8+ T cells and protection against viral challenge elicited by the AdC68 vaccines is not strongly inhibited in animals preimmune to AdHu5 virus. However, the response elicited by the AdHu5 vaccine is greatly attenuated in AdHu5 preimmune animals.

Using adoptive transfer models we determined that an adeno-associated viral vector of serotype 2 (AAV2) induces in mice proliferation of CD8(+) T cells that recognize an epitope within the viral capsid. Proliferation to an endogenous epitope within viral protein (VP)3 could be observed for at least 3 weeks while a foreign epitope placed at multiple copies within VP2 elicited CD8(+) T cell expansion for at least 10 weeks. These data show that capsid antigens of AAV2 degrade slowly over a period of weeks and during this period provide targets to CD8(+) T cells.

Despite enormous efforts by the scientific community, an effective HIV vaccine remains elusive. To further address to what degree T cells in absence of antibodies may protect against simian immunodeficiency virus (SIV) disease progression, rhesus macaques were vaccinated intramuscularly with a chimpanzee-derived Ad vector (AdC) serotype 6 and then boosted intramuscularly with a serologically distinct AdC vector of serotype 7 both expressing Gag of SIVmac239. Animals were subsequently boosted intramuscularly with a modified vaccinia Ankara (MVA) virus expressing Gag and Tat of the homologous SIV before mucosal challenge with a high dose of SIVmac239 given rectally. Whereas vaccinated animals showed only a modest reduction of viral loads, their overall survival was improved, in association with a substantial protection from the loss of CD4(+) T cells. In addition, the two vaccinated Mamu-A*01(+) macaques controlled viral loads to levels below detection within weeks after challenge. These data strongly suggest that T cells, while unable to affect SIV acquisition upon high-dose rectal infection, can reduce disease progression. Induction of potent T-cell responses should thus remain a component of our efforts to develop an efficacious vaccine to HIV-1.

Most studies on E1-deleted adenovirus (Ad) vectors as vaccine carriers for antigens of HIV-1 have focused on induction of central immune responses, although stimulation of mucosal immunity at the genital tract (GT), the primary port of entry of HIV-1, would also be highly desirable. In this study, different immunization protocols using chimpanzee-derived adenoviral (AdC) vectors expressing Gag of HIV-1 clade B given in heterologous prime-boost regimens were tested for induction of systemic and genital immune responses. Although i.n. immunization stimulated CD8(+) T-cell responses that could be detected in the GT, this route induced only marginal cellular responses in systemic tissues and furthermore numbers of Gag-specific CD8(+) T cells contracted sharply within a few weeks. On the contrary, i.m. immunization induced higher and more sustained frequencies of vaccine-induced cells which could be detected in the GT as well as systemic compartments. Antigen-specific CD8(+) T cells could be detected 1 year after immunization in all compartments analyzed. Genital memory cells secreted IFN-γ, expressed high levels of CD103 and their phenotypes were consistent with a state of activation. Taken together, the results presented here show that i.m. vaccination with chimpanzee-derived (simian) adenovirus vectors is a suitable strategy to induce a long-lived genital CD8(+) T-cell response.

Adenoviral vectors have shown great promise as vaccine carriers and in gene transfer to correct underlying genetic diseases. Traditionally, construction of adenoviral vectors is complex and time consuming. In this paper, we provide an improved method for efficient generation of novel adenoviral vectors by using direct cloning. We introduce a feasible and detailed protocol for the development of chimpanzee adenoviruses (Ads) as molecular clones, as well as for the generation of recombinant virus from the molecular clones. Recombinant viruses are genetically stable and induce potent immune responses in animals. Generation of new Ad molecular clones or new recombinant Ad can be achieved in 2 months or 2 weeks, respectively.

Immune responses diminish with age resulting in an increased susceptibility of the elderly to infectious agents and an inability to mount protective immune responses to vaccines. Immunosenescence affects multiple aspects of the immune system, including CD8+ T cells, which control viral infections and are assumed to prevent the development of cancers. In this study, we tested if CD8+ T cell responses in aged mice could be enhanced through a vaccine that concomitantly expresses Ag and a molecule that blocks an immunoinhibitory pathway. Specifically, we tested a vaccine based on a replication-defective chimpanzee-derived adenovirus vector expressing the nucleoprotein (NP) of influenza A virus as a fusion protein with the HSV type 1 glycoprotein D, which through binding to the herpes virus entry mediator, blocks the immunoinhibitory herpes virus entry mediator B and T lymphocyte attenuator/CD160 pathways. Our results show that the vaccine expressing a fusion protein of NP and glycoprotein D induces significantly higher NP-specific CD8+ T cell responses in young and aged mice compared with the vaccine expressing NP only.

Hepatic adeno-associated virus (AAV)-serotype 2-mediated gene transfer results in sustained transgene expression in experimental animals but not in human subjects. We hypothesized that loss of transgene expression in humans might be caused by immune memory mechanisms that become reactivated upon AAV vector transfer. Here, we tested the effect of immunological memory to AAV capsid on AAV-mediated gene transfer in a mouse model. Upon hepatic transfer of an AAV2 vector expressing human factor IX (hF.IX), mice immunized with adenovirus (Ad) vectors expressing AAV8 capsid before AAV2 transfer developed less circulating hF.IX and showed a gradual loss of hF.IX gene copies in liver cells as compared to control animals. This was not observed in mice immunized with an Ad vectors expressing AAV2 capsid before transfer of rAAV8-hF.IX vectors. The lower hF.IX expression was primarily linked to AAV-binding antibodies that lacked AAV-neutralizing activity in vitro rather than to AAV capsid-specific CD8(+) T cells.

In the phase IIb STEP trial an HIV-1 vaccine based on adenovirus (Ad) vectors of the human serotype 5 (AdHu5) not only failed to induce protection but also increased susceptibility to HIV-1 infection in individuals with preexisting neutralizing Abs against AdHu5. The mechanisms underlying the increased HIV-1 acquisition rates have not yet been elucidated. Furthermore, it remains unclear if the lack of the vaccine’s efficacy reflects a failure of the concept of T cell-mediated protection against HIV-1 or a product failure of the vaccine. Here, we compared two vaccine regimens based on sequential use of AdHu5 vectors or two different chimpanzee-derived Ad vectors in rhesus macaques that were AdHu5 seropositive or seronegative at the onset of vaccination. Our results show that heterologous booster immunizations with the chimpanzee-derived Ad vectors induced higher T and B cell responses than did repeated immunizations with the AdHu5 vector, especially in AdHu5-preexposed macaques.

The goal of these studies was to test whether adeno-associated virus (AAV) capsid-specific CD8(+) T cells cause loss of hepatic AAV-mediated gene expression in experimental animals. Mice immunized with adenoviral vectors expressing AAV capsid or with AAV vectors developed CD8(+) T cells in blood, lymphatic tissues, and liver to epitopes shared between AAV2 and AAV8, and serotype-specific neutralizing antibodies. At the height of the T cells' effector phase, mice were infused with a heterologous AAV vector expressing human factor IX under a hepatocyte-specific promoter. Despite the presence of lytic CD8(+) T cells in the liver, hepatic Factor IX expression was sustained and comparable in AAV-preimmune and naïve animals. These results suggest that, in mice, pre-existing CD8(+) T cells to AAV capsid do not affect the longevity of AAV-mediated hepatic gene transfer. These results are in contrast to the outcome of a recent gene therapy trial of hemophilia B patients who were treated by hepatic gene transfer of AAV2 vectors expressing Factor IX. The loss of Factor IX expression, accompanied by a rise in liver enzymes and detectable frequencies of circulating AAV capsid-specific T cells, suggested T-cell-mediated destruction of transduced hepatocytes following reactivation of AAV-specific T cells upon AAV transfer.

Recent studies have indicated that type I interferon (IFN) enhances antibody responses and promotes isotype switching. In this study, we analyzed the role of type I IFN signaling during the generation of transgene product-specific antibody responses elicited by recombinant adenovirus (Ad) vectors. A vector derived from a human Ad serotype (AdHu5) induced low levels of type I IFN following infection of dendritic cells (DCs) and stimulated normal transgene product-specific antibody responses in mice that have a defective type I IFN receptor (IFNAR(-/-)). A vector derived from a chimpanzee Ad serotype (AdC68) induced very high levels of type I IFN following infection of DCs, and surprisingly, primed stronger transgene product-specific antibody responses in IFNAR(-/-) mice compared to wild-type mice. The increased antibody response in IFNAR(-/-) mice vaccinated with the AdC68 vector was mainly due to the generation of IgG1 antibodies that were not elicited in wild-type mice. The induction of IgG1 antibodies correlated with an increase in transgene product expression in IFNAR(-/-) mice and was not associated with an increase in T helper 2 responses. We conclude that type I IFN, when induced at high levels, can downregulate transgene product expression of Ad vectors and inhibit the formation of optimal antibody responses.

DNA vaccines expressing the E6 or E7 oncoproteins of human papilloma virus type 16 (HPV-16) in either their wild-type form or fused to sequences that affect intracellular trafficking were tested for induction of protective immunity against tumor cell challenge in two models based on BALB/c and C57Bl/6 mice. The DNA vaccines to E7 gave uniformly disappointing results, while the DNA vaccine that expressed E6 linked to a viral leader sequence protected BALB/c mice against tumor cell challenge given before or after vaccination. The efficacy of this vaccine could be enhanced by a DNA vector prime/viral vector boost regimen. In contrast, priming of mice with the DNA vaccines to E7 reduced the efficacy of a viral vector expressing the same antigen.

Different vaccine constructs based on DNA vaccines and viral recombinant vaccines expressing mouse p53 were compared for induction of protective immune responses to challenge with a sarcoma cell line that expresses high levels of mutated p53 protein. Viral recombinant vaccines based on E1-deleted adenovirus or vaccinia virus recombinants expressing p53 with wild-type sequences in the mutational hotspot domain and a single mutation in the tetramerization domain (p53(mu338)) failed to induce protection against progression of this tumor cell line. A DNA vaccine expressing a form of p53 carrying the same point mutations as the tumor cell line showed low efficacy that was comparable to that of a DNA vaccine expressing p53(mu338). Efficacy of the DNA vaccine was augmented upon expressing p53(mu338) as a fusion protein linked to a viral leader sequence. Other modifications such as fusion to the signal sequence of the lysosome-associated membrane protein (LAMP) or ubiquitin failed to improve the efficacy of the vaccine to p53. Protection mediated by CD4(+) and CD8(+) T cells was specific for p53.

Summary How tumor-infiltrating T lymphocytes (TILs) adapt to the metabolic constrains within the tumor microenvironment (TME) and to what degree this affects their ability to combat tumor progression remain poorly understood. Using mouse melanoma models, we report that CD8 + TILs enhance peroxisome proliferator-activated receptor (PPAR)-α signaling and catabolism of fatty acids (FAs) when simultaneously subjected to hypoglycemia and hypoxia. This metabolic switch partially preserves CD8 + TILs' effector functions, although co-inhibitor expression increases during tumor progression regardless of CD8 + TILs' antigen specificity. Further promoting FA catabolism improves the CD8 + TILs' ability to slow tumor progression. PD-1 blockade delays tumor growth without changing TIL metabolism or functions. It synergizes with metabolic reprogramming of T cells to achieve superior antitumor efficacy and even complete cures.

Generation of three monoclonal antibodies (MAbs) to the major oncoproteins of human papillomavirus (HPV) was accomplished by an intense prime/boost regimen. Mice were primed with expression vectors expressing either the E6 or E7 oncoproteins of HPV-16 followed by boosting with a vaccinia virus construct and a replication-defective E1-deleted adenoviral recombinant of the human strain 5, and last, with baculovirus-derived HPV-16 E6 and E7 proteins in incomplete Freunds' adjuvant. Splenocytes were then fused with a myeloma cell line. The vaccination protocol generated one anti-E7 MAb of the IgM isotype and two anti-E6 MAbs of the IgG1 subisotype. The MAbs were tested for functionality in standard laboratory assays and found to detect the E6 and E7 proteins, respectively. The E7 MAb cross-reacted with the HPV-1a E7 oncoprotein. The binding sites of the MAbs were mapped to defined regions of each viral protein.

Most cancerous lesions of the uterine cervix are linked to persistent infections with human papillomaviruses (HPV), most notably HPV-16 or -18. Vaccine-induced immune responses to the HPV early antigens E6 and E7, which contribute to cell transformation and are thus expressed in these cervical cancers, could potentially eradicate malignant cells. We generated recombinant vaccines based on E1-deleted adenovirus human strain 5 or on vaccinia virus strain Copenhagen expressing either the E6 or E7 oncoproteins of HPV-16. The different vaccines were compared in two experimental mouse tumor models employing Balb/c or C57Bl/6 mice. Data presented here demonstrate that depending on the model either CD4+ or CD8+ T cells provide protection to tumor cell challenge, resulting in striking differences in the efficacy of the four vaccines under investigation.

The effect of palindromic CpG sequences on the B cell response to plasmid vectors expressing a highly immunogenic viral glycoprotein was investigated. Methylation of the CpG sequences of bacterial expression vectors abolished their ability to induce an antibody response to the transgene product in mice. The antibody response could be rescued by concomitant injection of oligonucleotides carrying immunostimulatory sequences. The B cell response to two plasmid vectors, both expressing the same viral glycoprotein but containing a different content of the highly stimulatory AACGTT motif, was compared. Comparable B cell responses were induced to the two constructs given at an optimal vaccine dose while the vector containing additional palindromic sequences resulted in higher antibody titers at a suboptimal dose. These data indicate that deletion of CpG motifs or methylation of such sequences in plasmid DNA can abrogate the immune response to the vector encoded antigen and might thus enhance their usefulness as gene therapy vehicles.

Vaccines that aim to expand tumor-specific CD8(+) T cells have yielded disappointing results in cancer patients although they showed efficacy in transplantable tumor mouse models. Using a system that more faithfully mimics a progressing cancer and its immunoinhibitory microenvironment, we here show that in transgenic mice, which gradually develop adenocarcinomas due to expression of HPV-16 E7 within their thyroid, a highly immunogenic vaccine expressing E7 only induces low E7-specific CD8(+) T-cell responses, which fail to affect the size of the tumors. In contrast, the same type of vaccine expressing E7 fused to herpes simplex virus (HSV)-1 glycoprotein D (gD), an antagonist of the coinhibitory B- and T-lymphocyte attenuator (BTLA)/CD160-herpes virus entry mediator (HVEM) pathways, stimulates potent E7-specific CD8(+) T-cell responses, which can be augmented by repeated vaccination, resulting in initial regression of even large tumor masses in all mice with sustained regression in more than half of them. These results indicate that active immunization concomitantly with blockade of the immunoinhibitory HVEM-BTLA/CD160 pathways through HSV-1 gD may result in sustained tumor regression.

In this study we compared a prime-boost regimen with two serologically distinct replication-defective adenovirus (Ad) vectors derived from chimpanzee serotypes C68 and C1 expressing Gag, Pol, gp140, and Nef of human immunodeficiency virus type 1 with a regimen in which replication-defective Ad vectors of the human serotype 5 (AdHu5) were given twice. Experiments were conducted in rhesus macaques that had or had not been preexposed to antigens of AdHu5. There was no significant difference in T-cell responses tested from peripheral blood of the different groups, although responses were overall highest in nonpreexposed animals immunized with the chimpanzee Ad vectors. Preexisting immunity to AdHu5 completely inhibited induction of transgene product-specific antibodies by the AdHu5 vectors without affecting antibody responses to the chimpanzee vectors. Upon euthanasia, T-cell responses were tested from a number of tissues. Preexisting immunity to AdHu5, commonly found in humans, changed the homing pattern of vaccine-induced T cells. In AdHu5-preexposed animals vaccinated with the chimpanzee Ad vectors, frequencies of transgene-specific T cells were higher in spleens than in blood, and in most preexposed animals vaccinated either with AdHu5 vectors or chimpanzee adenovirus vectors, frequencies of such T cells were exceptionally high in livers. The latter results indicate that analysis of T-cell responses solely from blood mononuclear cells of vaccine recipients may not suffice to compare the potencies of different vaccine regimens.

The 5-year survival rate of late-stage melanoma patients is less than 5%. Adoptive immune transfer has demonstrated the potency of melanoma-associated antigen (MAA)-specific T cells in inducing clinical responses. Nevertheless, cancer vaccines against melanoma that aim to induce such T cells have largely been ineffective in patients with advanced disease. One major barrier of cancer vaccine efficacy is immunoinhibition within the tumor microenvironment (TME). Vaccine-induced MAA-specific tumor infiltrating T cells (TILs) commonly show signs of T cell exhaustion within TME by upregulating co-inhibitory molecules combined with loss of functions. This has traditionally been viewed as the result of chronic antigen stimulation. However, our data show that TILs directed to an antigen that is not expressed by the tumor also become exhausted, which led us to hypothesize that exhaustion of TILs is linked to metabolic stress within the TME. Upon activation the energy production of T cells switches from oxidative phosphorylation (OXPHOS) to glycolysis, which requires enhanced uptake of glucose for ATP production. Glycolysis is also used by tumor cells, which may lead to glucose deprivation within the TME, forcing MAA-specific TILs to rely on OXPHOS. OXPHOS requires oxygen, which due to lack of blood supply can also be limiting within TME. TILs therefore face dual metabolic jeopardy, i.e., lack of glucose and oxygen, which may affect their functions and thereby impair the efficacy of cancer vaccines. Using a B16 mouse melanoma model and an adenoviral vector based polyepitope melanoma vaccine, we tested if vaccine-induced TILs experience metabolic stress within the TME and whether this contributes to T cell exhaustion. Our results indicate that tumors recruit vaccine-induced tyrosinase-related protein (Trp)-1-specific CD8+ T cells, which proliferate within TME, upregulate co-inhibitory markers and become functionally impaired. Activated CD8+ T cells directed to an unrelated antigen expressed by another vaccine were also recruited to the tumors; although they fail to proliferation they also increased co-inhibitors programmed death (PD)-1 and lymphocyte activation gene (LAG)-3 expression with downregulation of T-bet, a critical transcription factor regulating T cell effector functions. Comparing markers of mitochondrial functions and oxidative deficiencies between TILs and CD8+ T cells stimulated in vitro under conditions producing energy through OXPHOS or glycolysis and under normal or reduced oxygen levels, we showed that activated CD8+ TILs undergo oxidative stress evidenced by loss of mitochondrial membrane potential (MMP), increases of mitochondrial reactive oxygen species (MROS) and enhanced levels of hypoxia inducible factor 1 subunit α (HIF-1α) and its downstream target glucose transporter (Glut)1. This metabolic profile was largely mirrored by T cells activated in vitro under hypoxia. Furthermore, our data demonstrated links between the T cells metabolic stress and their exhaustion status. Enhanced expression of PD-1 was linked to lack of glucose as cells cultured in galactose significantly increased PD-1 compared to cells with access to glucose. Increased expression of LAG-3 was most pronounced on cells cultured under hypoxia or in medium with drug that stabilizes HIF-1α. Knocking down HIF-1α in activated CD8+ T cells cultured under hypoxia significantly reduced their LAG-3 expression and increased their T-bet levels, implying a direct impact of hypoxia-induced HIF-1α on the expression of co-inhibitor LAG-3 and overall exhaustion status of T cells. In summary, our data indicate that within the TME metabolic stress due to lack of glucose and oxygen rather than sustained activation causes T cell exhaustion. These findings suggest that lifting the metabolic stress of MAA-specific TILs may improve the therapeutic efficacy of cancer vaccines. Citation Format: Ying Zhang, Xiangyang Zhou, Raj Kurupati, Flavia Filisio, Abeer Hudaihed, Wynetta Giles-Davis, Hildegund CJ Ertl. Metabolic stress within tumors lead to exhaustion of melanoma antigen-specific CD8+ T cells. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B49.

Vaccines based on replication-defective adenoviral vectors are being developed for infectious agents and tumor-associated antigens. Early work focused on vaccines derived from a common human serotype of adenovirus, that is, adenovirus of the serotype 5 (AdHu5). Neutralizing antibodies against AdHu5 virus, present in a large percentage of the human population, dampen the efficacy of vaccines based on this carrier. To circumvent this problem, we generated vectors derived from chimpanzee adenoviruses. Here we describe some basic parameters of vectors derived from chimpanzee adenoviruses C68 and C7, including growth characteristics, yields of infectious particles, effects of additional deletions in E3 and E4 and lengths of the inserted foreign sequence as they relate to the suitability for their eventual development as vaccine carriers for clinical use.

To understand how vaccines initiate adaptive immune responses, it is necessary to study how they interact with APCs such as dendritic cells (DCs). In this study, we analyzed interactions between recombinant adenovirus (Ad) vectors and mouse DCs. Mouse bone marrow-derived DCs transduced with Ad vectors produced type I IFN, which promoted the maturation of both transduced and bystander DCs. DCs transduced with a vector derived from a chimpanzee Ad serotype (AdC68) produced more type I IFN and matured more efficiently compared with DCs transduced with a vector derived from a human Ad serotype (AdHu5). Both vectors stimulated type I IFN production independently of viral transcription, replication, and TLR signaling. However, each vector induced type I IFN through distinct pathways; whereas AdHu5 vectors required phosphoinositide-3-OH kinase for type I IFN induction, AdC68 vectors did not. Both vectors induced strong transgene product-specific CD8+ T cell responses in wild-type mice. DCs isolated from mice that have a defect in type I IFN signaling failed to undergo full maturation after Ad vaccination, but surprisingly, these mice mounted strong transgene product-specific CD8+ T cell responses. In these mice, we were able to detect a small number of transduced DCs that expressed high levels of costimulatory molecules, and these DCs were able to stimulate transgene product-specific CD8+ T cells. Thus, type I IFN signaling is an important component of Ad-mediated DC maturation but is dispensable during the generation of transgene product-specific CD8+ T cell responses.

Data presented here demonstrate that vaccine-induced CD8(+) T cells can eliminate their specific tumor-target with a two-staged attack. First, they release interferon-gamma that results in growth arrest of the tumor cells via induction of antiangiogenic mediators. Then, during the latter stages of the immune response, CD8(+) effector T cells eradicate the remaining tumor cells through perforin-mediated lysis. A combination of these two mechanisms is highly effective in the described model, while either pathway alone fails to completely achieve tumor rejection.

E1-deleted adenoviral recombinants most commonly based on the human serotype 5 (AdHu5) have been shown thus far to induce unsurpassed transgene product-specific CD8+ T cell responses. A large percentage of the adult human population carries neutralizing Abs due to natural exposures to AdHu5 virus. To circumvent reduction of the efficacy of adenovirus (Ad) vector-based vaccines by neutralizing Abs to the vaccine carrier, we developed E1-deleted adenoviral vaccine carriers based on simian serotypes. One of these carriers, termed AdC68, expressing a codon-optimized truncated form of gag of HIV-1 was shown previously to induce a potent transgene product-specific CD8+ T cell response in mice. We constructed a second chimpanzee adenovirus vaccine vector, termed AdC6, also expressing the truncated gag of HIV-1. This vector, which belongs to a different serotype than the AdC68 virus, induces high frequencies of gag-specific CD8+ T cells in mice including those pre-exposed to AdHu5 virus. Generation of an additional E1-deleted adenoviral vector of chimpanzee origin allows for sequential booster immunizations with heterologous vaccine carriers. In this study, we show that such heterologous prime boost regimens based on E1-deleted adenoviral vectors of different serotypes expressing the same transgene product are highly efficient in increasing the transgene product-specific CD8+ T cell response. They are equivalent to sequential vaccinations with an E1-deleted Ad vector followed by booster immunization with a poxvirus vector and they surpass regimens based on DNA vaccine prime followed by a recombinant adenoviral vector boost.

The three major concerns in the methodology of using overlapping synthetic peptides in solution to identify T-cell epitopes of proteins are the extended time required for the assembly and purification of individual peptides, the expenses involved, and the purity of the antigens when using high speed techniques. Many ELISA protocols take advantage of immobilized antigens for the detection of antibodies, and as an extension of this technique, analysis of cellulose membrane-bound B-cell epitopes is currently considered of high utility [1]. These peptide arrays are usually made by the SPOT synthesis methodology [2].

A method was developed to compare the lymphocytic infiltrates in regressing vs. progressing experimental mouse tumors using a model for human papillomavirus-16 (HPV-16) oncoprotein-linked cancer. Tumor cells mixed with matrigel, composed of natural matrix substances that provide a basement membrane structure for adherent cells, were inoculated into mice vaccinated with an efficacious vaccine to the E7 oncoprotein or a vaccine to a control antigen. The tumor cells remained within the solidified gel and recruited a cellular infiltrate that could readily be analyzed upon removal of the gelatinous mass containing progressing or regressing tumors. The results show that tumors recruit activated CD8 + T cells regardless of their antigen specificity. In regressing tumors expressing an appropriate target antigen for the vaccine-induced CD8 + T cells, a strong increase of the tumor antigen-specific T cell population was observed over time. Progressing tumors that lacked the target antigen for the activated CD8 + T cell population did not show this selective enrichment.

A novel method for quantitative analysis of tumor-specific CD8(+) T lymphocytes was developed. Lymphocytes from mice vaccinated with tumor-associated antigens (TAAs) were expanded for 5 days in tissue culture and then stimulated in vitro for 5 h with tumor cells. They were subsequently surface-stained for CD8 and for intracellular interferon gamma (IFN-gamma) and analyzed by flow cytometry. The specificity and sensitivity of this assay, staining of antigen-activated lymphocytes (SAAL), was comparable to that of surface staining with major histocompatibily class (MHC) I-peptide tetramers or of staining of peptide re-stimulated CD8(+) T cells for intracellular IFN-gamma. The assay did not exhibit the high background activity of traditional 51Cr-release assays that without elaborate effector cell purifications commonly fail to distinguish between T cell-mediated antigen-specific cytolysis and non-specific lysis by lymphokine-activated killer (LAK) cells. The described method, which does not require prior identification of individual TAAs and their T cell epitopes nor access to specific reagents such as MHC-peptide tetramers, represents a simple yet useful technique for studying tumor-specific cytolytic T cell responses.

Many enzyme-linked immunosorbent assays take advantage of immobilized antigens for the identification of antibody binding sites. Generally, the analysis of cellulose membrane-bound B-cell epitopes is currently considered of high utility. We adapted this methodology for the stimulation of a T helper cell hybridoma with known specificity. Forty overlapping peptides corresponding to the entire rabies virus nucleoprotein were synthesized in duplicates on a single sheet of 90×130 mm size amino-modified paper. The efficacy of the peptide assembly was monitored by color staining of the unreacted amino groups. After completion of the synthesis, the side-chain protecting groups were removed, and the membrane was thoroughly cleaned of all organic and inorganic contaminants. The membrane was cut into pieces, and a standard lymphokine release assay was performed directly from the paper-bound antigens. From all the 40 peptide spots only peptide 31D stimulated the proliferation of the 9C5.D8-H T-cell hybridoma, known to react to this peptide. By using this protocol, as little as 0.4 μg (approximately 200 pmole) of peptide could be detected. According to mass spectrometry the T-cell stimulation proceeded as a true solid-phase assay. The peptide neither leached from the membrane nor was cleaved by the medium–splenocyte mixture. Additionally, tryptic digestion of the cellulose membrane released the expected peptide fragments.