Susan A. Holechek

Vaccinia virus vaccines: Past, present and future

Vaccinia virus (VACV) has been used more extensively for human immunization than any other vaccine. For almost two centuries, VACV was employed to provide cross-protection against variola virus, the causative agent of smallpox, until the disease was eradicated in the late 1970s. Since that time, continued research on VACV has produced a number of modified vaccines with improved safety profiles. Attenuation has been achieved through several strategies, including sequential passage in an alternative host, deletion of specific genes or genetic engineering of viral genes encoding immunomodulatory proteins. Some highly attenuated third- and fourth-generation VACV vaccines are now being considered for stockpiling against a possible re-introduction of smallpox through bioterrorism. Researchers have also taken advantage of the ability of the VACV genome to accommodate additional genetic material to produce novel vaccines against a wide variety of infectious agents, including a recombinant VACV encoding the rabies virus glycoprotein that is administered orally to wild animals. This review provides an in-depth examination of these successive generations of VACV vaccines, focusing on how the understanding of poxviral replication and viral gene function permits the deliberate modification of VACV immunogenicity and virulence.

Enhanced T-Cell Immunity to Osteosarcoma Through Antibody Blockade of PD-1/PD-L1 Interactions

Osteosarcoma is the most common bone cancer in children and adolescents. Although 70% of patients with localized disease are cured with chemotherapy and surgical resection, patients with metastatic osteosarcoma are typically refractory to treatment. Numerous lines of evidence suggest that cytotoxic T lymphocytes (CTLs) limit the development of metastatic osteosarcoma. We have investigated the role of PD-1, an inhibitory TNFR family protein expressed on CTLs, in limiting the efficacy of immune-mediated control of metastatic osteosarcoma. We show that human metastatic, but not primary, osteosarcoma tumors express a ligand for PD-1 (PD-L1) and that tumor-infiltrating CTLs express PD-1, suggesting this pathway may limit CTLs control of metastatic osteosarcoma in patients. PD-L1 is also expressed on the K7M2 osteosarcoma tumor cell line that establishes metastases in mice, and PD-1 is expressed on tumor-infiltrating CTLs during disease progression. Blockade of PD-1/PD-L1 interactions dramatically improves the function of osteosarcoma-reactive CTLs in vitro and in vivo, and results in decreased tumor burden and increased survival in the K7M2 mouse model of metastatic osteosarcoma. Our results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma should be pursued as a therapeutic strategy.

Improved NYVAC-Based Vaccine Vectors

While as yet there is no vaccine against HIV/AIDS, the results of the phase III Thai trial (RV144) have been encouraging and suggest that further improvements of the prime/boost vaccine combination of a poxvirus and protein are needed. With this aim, in this investigation we have generated derivatives of the candidate vaccinia virus vaccine vector NYVAC with potentially improved functions. This has been achieved by the re-incorporation into the virus genome of two host range genes, K1L and C7L, in conjunction with the removal of the immunomodulatory viral molecule B19, an antagonist of type I interferon action. These novel virus vectors, referred to as NYVAC-C-KC and NYVAC-C-KC-ΔB19R, have acquired relevant biological characteristics, giving higher levels of antigen expression in infected cells, replication-competency in human keratinocytes and dermal fibroblasts, activation of selective host cell signal transduction pathways, and limited virus spread in tissues. Importantly, these replication-competent viruses have been demonstrated to maintain a highly attenuated phenotype.

Role of short-term dispersal on the dynamics of Zika virus in an extreme idealized environment

In November 2015, El Salvador reported their first case of Zika virus (ZIKV) infection, an event followed by an explosive outbreak that generated over 6000 suspected cases in a period of two months. National agencies began implementing control measures that included vector control and recommending an increased use of repellents. Further, in response to the alarming and growing number of microcephaly cases in Brazil, the importance of avoiding pregnancies for two years was stressed. In this paper, we explore the role of mobility within communities characterized by extreme poverty, crime and violence. Specifically, the role of short term mobility between two idealized interconnected highly distinct communities is explored in the context of ZIKV outbreaks. We make use of a Lagrangian modeling approach within a two-patch setting in order to highlight the possible effects that short-term mobility, within highly distinct environments, may have on the dynamics of ZIKV outbreak when the overall goal is to reduce the number of cases not just in the most affluent areas but everywhere. Outcomes depend on existing mobility patterns, levels of disease risk, and the ability of federal or state public health services to invest in resource limited areas, particularly in those where violence is systemic. The results of simulations in highly polarized and simplified scenarios are used to assess the role of mobility. It quickly became evident that matching observed patterns of ZIKV outbreaks could not be captured without incorporating increasing levels of heterogeneity. The number of distinct patches and variations on patch connectivity structure required to match ZIKV patterns could not be met within the highly aggregated model that is used in the simulations.

The attenuated NYCBH vaccinia virus deleted for the immune evasion gene, E3L, completely protects mice against heterologous challenge with ectromelia virus

The New York City Board of Health (NYCBH) vaccinia virus (VACV) vaccine strain was deleted for the immune evasion gene, E3L, and tested for its pathogenicity and ability to protect mice from heterologous challenge with ectromelia virus (ECTV). NYCBHΔE3L was found to be highly attenuated for pathogenicity in a newborn mouse model and showed a similar attenuated phenotype as the NYVAC strain of vaccinia virus. Scarification with one or two doses of the attenuated NYCBHΔE3L was able to protect mice equally as well as NYCBH from death, weight loss, and viral spread to visceral organs. A single dose of NYCBHΔE3L resulted in low poxvirus-specific antibodies, and a second dose increased levels of poxvirus-specific antibodies to a level similar to that seen in animals vaccinated with a single dose of NYCBH. However, similar neutralizing antibody titers were observed following one or two doses of NYCBHΔE3L or NYCBH. Thus, NYCBHΔE3L shows potential as a candidate for a safer human smallpox vaccine since it protects mice from challenge with a heterologous poxvirus.

On the dynamics of dengue virus type 2 with residence times and vertical transmission

A two-patch mathematical model of Dengue virus type 2 (DENV-2) that accounts for vectors’ vertical transmission and between patches human dispersal is introduced. Dispersal is modelled via a Lagrangian approach. A host-patch residence-times basic reproduction number is derived and conditions under which the disease dies out or persists are established. Analytical and numerical results highlight the role of hosts’ dispersal in mitigating or exacerbating disease dynamics. The framework is used to explore dengue dynamics using, as a starting point, the 2002 outbreak in the state of Colima, Mexico.

Vertical Transmission in a Two-Strain Model of Dengue Fever

Abstract The role of vertical transmission in vectors has rarely been addressed in the study of dengue dynamics and control, in part because it was not considered a critical population-level factor. In this paper, we apply the pioneering modeling ideas of Ross and MacDonald, motivated by the context of the 2000–2001 dengue outbreak in Peru, to assess the dynamics of multi-strain competition. An invading strain of dengue virus (DENV-2) from Asia rapidly circulated into Peru eventually displacing DENV-2 American. A host-dengue model that considers the competing dynamics of these two DENV-2 genotypes, the resident or the American type and the invasive more virulent Asian strain, is introduced and analyzed. The model incorporates vertical transmission by DENV-2 Asian a potentially advantageous trait. Conditions for competitive exclusion of dengue strains are established. The model is used to show that lower transmission rates of DENV-2 Asian are sufficient for displacing DENV-2 American in the presence of vertical transmission.

Use of a Recombinant Vaccinia Virus Expressing Interferon Gamma for Post-Exposure Protection against Vaccinia and Ectromelia Viruses

Post-exposure vaccination with vaccinia virus (VACV) has been suggested to be effective in minimizing death if administered within four days of smallpox exposure. While there is anecdotal evidence for efficacy of post-exposure vaccination this has not been definitively studied in humans. In this study, we analyzed post-exposure prophylaxis using several attenuated recombinant VACV in a mouse model. A recombinant VACV expressing murine interferon gamma (IFN-γ) was most effective for post-exposure protection of mice infected with VACV and ectromelia virus (ECTV). Untreated animals infected with VACV exhibited severe weight loss and morbidity leading to 100% mortality by 8 to 10 days post-infection. Animals treated one day post-infection had milder symptoms, decreased weight loss and morbidity, and 100% survival. Treatment on days 2 or 3 post-infection resulted in 40% and 20% survival, respectively. Similar results were seen in ECTV-infected mice. Despite the differences in survival rates in the VACV model, the viral load was similar in both treated and untreated mice while treated mice displayed a high level of IFN-γ in the serum. These results suggest that protection provided by IFN-γ expressed by VACV may be mediated by its immunoregulatory activities rather than its antiviral effects. These results highlight the importance of IFN-γ as a modulator of the immune response for post-exposure prophylaxis and could be used potentially as another post-exposure prophylaxis tool to prevent morbidity following infection with smallpox and other orthopoxviruses.

Enhancement of T cell immunity to osteosarcoma through modulation of PD-1/PD-L1 interactions

Osteosarcomas remain one of the most commonly occurring bone cancers in adolescents, accounting for approximately 3% of all diagnosed childhood malignancies. Approximately 30% of osteosarcoma patients develop pulmonary metastatic disease, which is associated with poor prognosis, as 5-year survival rates for these patients are less than 20%. Cytotoxic T-lymphocytes (CTL) likely play an important role in the control of developing osteosarcomas as polymorphisms in cytotoxic T lymphocyte antigen 4 (CTLA-4), an inhibitory tumor necrosis factor receptor (TNFR) family member, result in increased CTLA-4 protein expression levels, and are associated with higher risk of developing osteosarcoma. While CTLA-4 is involved in limiting the generation of CTL responses, other inhibitory TNFR family members limit the function of CTL after tumor metastases and during disease progression. In particular, ligation of programmed death receptor-1 (PD-1), expressed on tumor-specific CTL, by PD-1 ligand (PD-L1) on tumors, results in potent inhibition of CTL proliferation, cytokine production, and cytotoxicity, leading to tumor progression. Using a mouse model of metastatic osteosarcoma, we have shown that PD-1 is expressed on osteosarcoma tumor infiltrating CTL, and that osteosarcoma cells express PD-L1. Human metastatic osteosarcomas also exhibit increased expression of PD-L1 compared to pre-metastatic tumors, with PD-1 expression on metastatic tumor infiltrating lymphocytes. Furthermore, we have shown that removal of PD-1/PD-L1 interactions in the mouse metastatic osteosarcoma models dramatically improves survival outcomes. These studies of the role of PD-1 in inhibition of CTL-mediated eradication in metastatic osteosarcoma in mouse models and in primary human metastatic osteosarcoma provide the necessary pre-clinical data required for subsequent clinical testing of therapeutic approaches to block PD-1/PD-L1 interactions for treatment of metastatic osteosarcoma patients.

OA021-01. Construction and characterization of replication competent attenuated NYVAC-based vectors as potential HIV vaccines

To decrease virulence, we deleted the E3L gene, which is required for interferon-resistance and virulence, and replaced it with a gene from Ambystoma tigrinum virus (ATV, the new virus is NYVAC-C+12-ATV), which restores a single round of replication. Results In vitro characterization of the constructs demonstrates restoration of replication in primary and human cell lines. NYVAC-C+12-ATV leads to induction of pro-inflammatory signal transduction pathways. Pathogenicity studies in newborn mice demonstrate attenuation of 3–5 logs when compared to wt vaccinia virus or to NYCBH.