Investigation of the Optimal Prime Boost Spacing Regimen for a Cancer Therapeutic Vaccine Targeting Human Papillomavirus.

Therapeutic vaccine studies should be designed to elicit durable, high magnitude, and efficacious T cell responses, all of which can be impacted by the choice of the vaccination schedule. Here, we compare different prime-boost intervals (PBI) in a human papillomavirus (HPV) model using a HPV16E7E6 Venezuelan equine encephalitis virus replicon particle (VRP) vaccination to address the optimal boosting schedule, quality of immune response, and overall in vivo efficacy. Six different vaccine regimens were tested with each group receiving booster vaccinations at different time intervals. Analysis of T-cell responses demonstrated a significant HPV16 E7 specific CD8+ T cell response with at minimum a one-week PBI between antigen re-exposure. Significant E7-specific in vivo cytotoxicity was also observed with longer PBIs. Additionally, longer PBIs led to an enhanced memory recall response to tumor challenge, which correlated with differential expansion of T cell memory subsets. Our findings imply that when using alphavirus vector platforms as a vaccination strategy, a one-week PBI is sufficient to induce high magnitude effector T cells with potent anti-tumor activity. However, longer PBIs lead to enhanced long-term protective anti-tumor immunity. These findings have implications for therapeutic vaccine clinical trials in which shorter intervals of prime-boost regimens may lead to suboptimal durable immune responses.

A comparative study between outbred and inbred rat strains for the use in in vivo IPV potency testing.

In vivo potency testing of inactivated poliovirus vaccines (IPV) is generally performed in rats, although no systematic investigation has identified the most appropriate rat strain for anti-poliovirus antibody quantification. We investigated humoral immune responses to IPV in five different rat strains to identify the most suitable strain. Three outbred (Wistar, Wistar Hannover, Sprague-Dawley) and two inbred rat strains (Fisher 344, Wistar Furth) were immunized intramuscularly with a full or one-fifth human dose of commercial IPV. Anti-poliovirus neutralizing antibody (NA) titers were measured using Salk and Sabin virus neutralizing assays. Post-vaccination responses varied between strains; inbred strains showed greater animal-to-animal variation in NA responses than outbred strains. Virus NA titers persisted for 9 weeks with little reduction in the response. The outbred Wistar rat model was identified as the preferred strain for IPV potency testing based on its capacity to produce high, dose-dependent anti-poliovirus NA responses, with low animal-to-animal variation.

Recombinant measles virus incorporating heterologous viral membrane proteins for use as vaccines.

Recombinant measles virus (rMV) vectors expressing heterologous viral membrane protein antigens are potentially useful as vaccines. Genes encoding the mumps virus haemagglutinin-neuraminidase (MuV-HN), the influenza virus haemagglutinin (Flu-HA) or the respiratory syncytial virus fusion (RSV-F) proteins were inserted into the genome of a live attenuated vaccine strain of measles virus. Additionally, in this case rMV with the MuV-HN or the influenza HA inserts, chimeric constructs were created that harboured the measles virus native haemagglutinin or fusion protein cytoplasmic domains. In all three cases, sucrose-gradient purified preparations of rMV were found to have incorporated the heterologous viral membrane protein on the viral membrane. The possible utility of rMV expressing RSV-F (rMV.RSV-F) as a vaccine was tested in a cotton rat challenge model. Vaccination with rMV.RSV-F efficiently induced neutralizing antibodies against RSV and protected animals from infection with RSV in the lungs.

A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism.

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infections and is the leading cause of infant hospitalizations. Recently, a promising vaccine antigen based on the RSV fusion protein (RSV F) stabilized in the native prefusion conformation has been described. Here we report alternative strategies to arrest RSV F in the prefusion conformation based on the prevention of hinge movements in the first refolding region and the elimination of proteolytic exposure of the fusion peptide. A limited number of unique mutations are identified that stabilize the prefusion conformation of RSV F and dramatically increase expression levels. This highly stable prefusion RSV F elicits neutralizing antibodies in cotton rats and induces complete protection against viral challenge. Moreover, the structural and biochemical analysis of the prefusion variants suggests a function for p27, the excised segment that precedes the fusion peptide in the polypeptide chain.

Recombinant low-seroprevalent adenoviral vectors Ad26 and Ad35 expressing the respiratory syncytial virus (RSV) fusion protein induce protective immunity against RSV infection in cotton rats.

RSV is an important cause of lower respiratory tract infections in children, the elderly and in those with underlying medical conditions. Although the high disease burden indicates an urgent need for a vaccine against RSV, no licensed RSV vaccine is currently available. We developed an RSV vaccine candidate based on the low-seroprevalent human adenovirus serotypes 26 and 35 (Ad26 and Ad35) encoding the RSV fusion (F) gene. Single immunization of mice with either one of these vectors induced high titers of RSV neutralizing antibodies and high levels of F specific interferon-gamma-producing T cells. A Th1-type immune response was indicated by a high IgG2a/IgG1 ratio of RSV-specific antibodies, strong induction of RSV-specific interferon-gamma and tumor necrosis factor-alpha cytokine producing CD8 Tcells, and low RSV-specific CD4 T-cell induction. Both humoral and cellular responses were increased upon a boost with RSV-F expressing heterologous adenovirus vector (Ad35 boost after Ad26 prime or vice versa). Both single immunization and prime-boost immunization of cotton rats induced high and long-lasting RSV neutralizing antibody titers and protective immunity against lung and nasal RSV A2 virus load up to at least 30 weeks after immunization. Cotton rats were also completely protected against challenge with a RSV B strain (B15/97) after heterologous prime-boost immunization. Lungs from vaccinated animals showed minimal damage or inflammatory infiltrates post-challenge, in contrast to animals vaccinated with formalin-inactivated virus. Our results suggest that recombinant human adenoviral Ad26 and Ad35 vectors encoding the RSV F gene have the potential to provide broad and durable protection against RSV in humans, and appear safe to be investigated in infants.

A novel murine model for evaluating bovine papillomavirus prophylactics/therapeutics for equine sarcoid-like tumours.

Equine sarcoids are highly recurrent bovine papillomavirus (BPV)-induced fibroblastic neoplasms that are the most common skin tumours in horses. In order to facilitate the study of potential equine sarcoid prophylactics or therapeutics, which can be a slow and costly process in equines, a murine model for BPV-1 protein-expressing equine sarcoid-like tumours was developed in mice through stable transfection of BPV-1 E5 and E6 in a murine fibroblast tumour cell line (K-BALB). Like equine sarcoids, these murine tumour cells (BPV-KB) were of fibroblast origin, were tumorigenic and expressed BPV-1 proteins. As an initial investigation of the preclinical potential of this tumour model for equine sarcoids prophylactics, mice were immunized with BPV-1 E5E6 Venezuelan equine encephalitis virus replicon particles, prior to BPV-KB challenge, which resulted in an increased tumour-free period compared with controls, indicating that the BPV-KB murine model may be a valuable preclinical alternative to equine clinical trials.

EcPV2 DNA in equine genital squamous cell carcinomas and normal genital mucosa.

Squamous cell carcinoma (SCC) represents the most common genital malignant tumor in horses. Similar to humans, papillomaviruses (PVs) have been proposed as etiological agents and recently Equine papillomavirus type 2 (EcPV2) has been identified in a subset of genital SCCs. The goals of this study were (1) to determine the prevalence of EcPV2 DNA in tissue samples from equine genital SCCs, penile intraepithelial neoplasia (PIN) and penile papillomas, using EcPV2-specific PCR, (2) to examine the prevalence of latent EcPV2 infection in healthy genital mucosa and (3) to determine genetic variability within EcPV2 and to disentangle phylogenetic relationships of EcPV2 among PVs. EcPV2 DNA was detected in all but one penile SCC (15/16), in all PIN lesions (8/8) and penile papillomas (4/4). Additionally, EcPV2 DNA was demonstrated in one of two metastasized lymph nodes, one contact metastasis in the mouth, two vaginal and one anal lesion. In healthy horses, EcPV2 DNA was detected in 10% (4/39) of penile swabs but in none of vulvovaginal swabs (0/20). This study confirms the presence of EcPV2 DNA in equine genital SCCs and shows its involvement in anal lesions, a lymph node and contact metastases. Latent EcPV2 presence was also shown in normal male genital mucosa. We found that different EcPV2 variants cocirculate among horses and that EcPV2 is related to the Delta+Zeta PVs and is only a very distant relative of high-risk human PVs causing genital cancer. Thus, similar viral tropism and similar malignant outcome of the infection do not imply close evolutionary relationship.

EcPV2 DNA in equine squamous cell carcinomas and normal genital and ocular mucosa.

Squamous cell carcinoma (SCC) represents the most common malignant tumour of the eye and external genitals in horses. Comparable to humans, papillomaviruses (PV) have been proposed as etiological agents of cancer in horses and recently, Equine papillomavirus type 2 (EcPV2) has been identified in genital SCCs. Hitherto it had never been demonstrated in ocular SCCs. The first goal of this study was to determine the prevalence of EcPV2 DNA in tissue samples from equine genital and ocular SCCs, genital papillomas and penile intraepithelial neoplasia (PIN) lesions, using EcPV2-specific PCR. The second goal was to investigate the possibility of latent EcPV2 infection in the genital and ocular mucosa of healthy horses on swabs obtained from the eye, penis, vulvovaginal region and cervix. EcPV2 DNA was detected in all genital SCCs (17/17), genital papillomas (8/8), PIN lesions (11/11) and ocular SCCs (9/9). In healthy horses, EcPV2 DNA was detected in 43% (17/40) of penile swabs, 53% (9/17) of vulvovaginal swabs, 47% (8/17) of cervical swabs and 57% (32/56) of ocular swabs. This study confirms the presence of EcPV2 DNA in equine genital SCCs. Moreover, we demonstrate for the first time its involvement in other genital lesions and in ocular SCCs and latent EcPV2 infections in normal genital (including cervical) and ocular equine mucosa. The close relatives of EcPV2 are associated to cutaneous lesions, and this virus is not related to high-risk human papillomaviruses causing cervical cancer. Thus, similar viral tropism does not imply close evolutionary relationship.

Bovine papillomavirus DNA can be detected in keratinocytes of equine sarcoid tumors.

Bovine papillomavirus (BPV)-1 and -2 is linked to equine sarcoids, a commonly observed skin tumor in horses that is of considerable veterinary importance. Previous studies using in situ hybridization have detected BPV DNA only in fibroblasts and not in keratinocytes of sarcoids. In contrast, normal equine skin latently infected with BPV shows a dysplastic epithelium without dermal changes, similar to lesions induced by other papillomavirus types infecting the epithelium. The first goal of our study was to describe the epidermal and dermal characteristics of several stages in sarcoid development. Next, we explored whether BPV can infect epidermal cells in the horse using real-time PCR on laser-micro-dissected keratinocytes and fibroblasts. We found that latently infected normal skin samples and a subset of early stage sarcoids show dysplastic, koilocyte-like epithelial changes. BPV DNA was detected in keratinocytes in 40% of the samples with these particular epithelial properties, whereas advanced sarcoids only had BPV DNA in the fibroblasts. These data may indicate a novel and intriguing pathway of BPV infection in the horse composed of a first step of keratinocyte infection, followed by migration of viral material towards the dermis resulting in infection of sub-epidermal fibroblasts and their fully transformed phenotype. Additionally, an example of co-existence of a dermal BPV-1 and an epidermal BPV-2 infection in the same lesion is shown, indicating that horses can harbor infection with more than one BPV type at the same time.

Expression of LIGHT/TNFSF14 combined with vaccination against human papillomavirus Type 16 E7 induces significant tumor regression.

LIGHT, a ligand for the lymphotoxin-beta receptor, establishes lymphoid-like tissues inside tumor sites and recruits naïve T cells into the tumor. However, whether these infiltrating T cells are specific for tumor antigens is not known. We hypothesized that therapy with LIGHT can expand functional tumor-specific CD8(+) T cells that can be boosted using HPV16E6E7-Venezuelan equine encephalitis virus replicon particles (HPV16-VRP) and that this combined therapy can eradicate human papillomavirus 16 (HPV16)-induced tumors. Our data show that forced expression of LIGHT in tumors results in an increase in expression of IFNgamma and chemoattractant cytokines such as interleukin-1a, MIG, and macrophage inflammatory protein-2 within the tumor and that this tumor microenvironment correlates with an increase in frequency of tumor-infiltrating CD8(+) T cells. Forced expression of LIGHT also results in the expansion of functional T cells that recognize multiple tumor antigens, including HPV16 E7, and these T cells prevent the outgrowth of tumors on secondary challenge. Subsequent boosting of E7-specific T cells by vaccination with HPV16-VRP significantly increases their frequency in both the periphery and the tumor and leads to the eradication of large well-established tumors, for which either treatment alone is not successful. These data establish the safety of Ad-LIGHT as a therapeutic intervention in preclinical studies and suggest that patients with HPV16(+) tumors may benefit from combined immunotherapy with LIGHT and antigen-specific vaccination.

Osteochondral fragmentation in the synovial pad of the fetlock in Warmblood horses.

OBJECTIVES: To determine clinical and arthroscopic characteristics associated with fragments in the synovial pad of the fetlock and to characterize their morphology. STUDY DESIGN: Retrospective study. ANIMALS: Warmblood horses (n=104) with fragment(s) in the synovial pad. METHODS: Signalment and results of radiographic and clinical examination were collected before surgery. After arthroscopic fragment removal and joint evaluation for synovial and/or cartilage abnormalities, fragments were measured, and evaluated by histopathology. RESULTS: Synovial pad fragments (n=142) were removed from 127 fetlocks. Two older horses had lameness. During arthroscopy, abnormalities were observed in 40 joints (31.5%) and multivariate logistic regression analysis showed a significant correlation between the observed arthroscopic abnormalities and the presence of large fragments (P=.016). Fragments were osteochondral bodies completely surrounded by fibrous tissue. At the edges of the hyaline cartilage cap an underlying fibrous structure was obvious in the extracellular matrix giving it a reactive pattern. CONCLUSIONS: Although the impact on lameness was minimal, there was a significant correlation between arthroscopic abnormalities and presence of large synovial pad fragments. On histopathology, these osteochondral fragments are embedded in fibrous tissue and show a reactive pattern. They are not a manifestation of any well-described joint pathology. CLINICAL RELEVANCE: Large synovial pad fragments in Warmblood horses can be associated with synovial and cartilage abnormalities, but further studies are warranted to determine their origin and clinical importance.

Selection of a set of reliable reference genes for quantitative real-time PCR in normal equine skin and in equine sarcoids.

BACKGROUND: Real-time quantitative PCR can be a very powerful and accurate technique to examine gene transcription patterns in different biological conditions. One of the critical steps in comparing transcription profiles is accurate normalisation. In most of the studies published on real-time PCR in horses, normalisation occurred against only one reference gene, usually GAPDH or ACTB, without validation of its expression stability. This might result in unreliable conclusions, because it has been demonstrated that the expression levels of so called "housekeeping genes" may vary considerably in different tissues, cell types or disease stages, particularly in clinical samples associated with malignant disease. The goal of this study was to establish a reliable set of reference genes for studies concerning normal equine skin and equine sarcoids, which are the most common skin tumour in horses. RESULTS: In the present study the gene transcription levels of 6 commonly used reference genes (ACTB, B2M, HPRT1, UBB, TUBA1 and RPL32) were determined in normal equine skin and in equine sarcoids. After applying the geNorm applet to this set of genes, TUBA1, ACTB and UBB were found to be most stable in normal skin and B2M, ACTB and UBB in equine sarcoids. CONCLUSION: Based on these results, TUBA1, ACTB and UBB, respectively B2M, ACTB and UBB can be proposed as reference gene panels for accurate normalisation of quantitative data for normal equine skin, respectively equine sarcoids. When normal skin and equine sarcoids are compared, the use of the geometric mean of UBB, ACTB and B2M can be recommended as a reliable and accurate normalisation factor.