Julia L. Hurwitz

Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients

T-cell immunotherapy that takes advantage of Epstein-Barr virus (EBV)-stimulated immunity has the potential to fill an important niche in targeted therapy for EBV-related cancers. To address questions of long-term efficacy, safety, and practicality, we studied 114 patients who had received infusions of EBV-specific cytotoxic T lymphocytes (CTLs) at 3 different centers to prevent or treat EBV(+) lymphoproliferative disease (LPD) arising after hematopoietic stem cell transplantation. Toxicity was minimal, consisting mainly of localized swelling at sites of responsive disease. None of the 101 patients who received CTL prophylaxis developed EBV(+) LPD, whereas 11 of 13 patients treated with CTLs for biopsy-proven or probable LPD achieved sustained complete remissions. The gene-marking component of this study enabled us to demonstrate the persistence of functional CTLs for up to 9 years. A preliminary analysis indicated that a patient-specific CTL line can be manufactured, tested, and infused for

Localization of CD4+ T cell epitope hotspots to exposed strands of HIV envelope glycoprotein suggests structural influences on antigen processing

6095, a cost that compares favorably with other modalities used in the treatment of LPD. We conclude that the CTL lines described here provide safe and effective prophylaxis or treatment for lymphoproliferative disease in transplantation recipients, and the manufacturing methodology is robust and can be transferred readily from one institution to another without loss of reproducibility.

Recombinant Sendai Virus Expressing the G Glycoprotein of Respiratory Syncytial Virus (RSV) Elicits Immune Protection against RSV

The spectrum of immunogenic epitopes presented by the H2-IAb MHC class II molecule to CD4+ T cells has been defined for two different (clade B and clade D) HIV envelope (gp140) glycoproteins. Hybridoma T cell lines were generated from mice immunized by a sequential prime and boost regime with DNA, recombinant vaccinia viruses, and protein. The epitopes recognized by reactive T cell hybridomas then were characterized with overlapping peptides synthesized to span the entire gp140 sequence. Evidence of clonality also was assessed with antibodies to T cell receptor Vα and Vβ chains. A total of 80 unique clonotypes were characterized from six individual mice. Immunogenic peptides were identified within only four regions of the HIV envelope. These epitope hotspots comprised relatively short sequences (≈20–80 aa in length) that were generally bordered by regions of heavy glycosylation. Analysis in the context of the gp120 crystal structure showed a pattern of uniform distribution to exposed, nonhelical strands of the protein. A likely explanation is that the physical location of the peptide within the native protein leads to differential antigen processing and consequent epitope selection.

Respiratory syncytial virus vaccine development

ABSTRACT Although RSV causes serious pediatric respiratory disease, an effective vaccine does not exist. To capture the strengths of a live virus vaccine, we have used the murine parainfluenza virus type 1 (Sendai virus [SV]) as a xenogeneic vector to deliver the G glycoprotein of RSV. It was previously shown (J. L. Hurwitz, K. F. Soike, M. Y. Sangster, A. Portner, R. E. Sealy, D. H. Dawson, and C. Coleclough, Vaccine 15:533-540, 1997) that intranasal SV protected African green monkeys from challenge with the related human parainfluenza virus type 1 (hPIV1), and SV has advanced to clinical trials as a vaccine for hPIV1 (K. S. Slobod, J. L. Shenep, J. Lujan-Zilbermann, K. Allison, B. Brown, R. A. Scroggs, A. Portner, C. Coleclough, and J. L. Hurwitz, Vaccine, in press). Recombinant SV expressing RSV G glycoprotein was prepared by using reverse genetics, and intranasal inoculation of cotton rats elicited RSV-specific antibody and elicited protection from RSV challenge. RSV G-recombinant SV is thus a promising live virus vaccine candidate for RSV.

A novel vaccine regimen utilizing DNA, vaccinia virus and protein immunizations for HIV-1 envelope presentation

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract viral disease in infants and young children. Presently, there are no explicit recommendations for RSV treatment apart from supportive care. The virus is therefore responsible for an estimated 160,000 deaths per year worldwide. Despite half a century of dedicated research, there remains no licensed vaccine product. Herein are described past and current efforts to harness innate and adaptive immune potentials to combat RSV. A plethora of candidate vaccine products and strategies are reviewed. The development of a successful RSV vaccine may ultimately stem from attention to historical lessons, in concert with an integral partnering of immunology and virology research fields.

Sendai virus-based RSV vaccine protects African green monkeys from RSV infection

Recombinant DNA and vaccinia virus (VV) vectors that express envelope (Env) proteins of the human immunodeficiency virus (HIV) have each been prominently utilized in vaccine development. These two vectors (termed DNA-Env and VV-Env) are attractive vaccine candidates due to their abilities to elicit both cytotoxic T-lymphocyte and B-cell responses. Our previous work demonstrated that DNA-Env primed animals, that were relatively unresponsive to DNA-Env boosters, could be immunized with VV-Env to yield more than a 100-fold increase in antibody responses. Here we show: (1) results with an optimized vaccine regimen that primes with DNA-Env, boosts with VV-Env, and re-boosts with purified Env proteins, (2) enhanced responses with 8 rather than 16 week intervals between VV-Env and protein immunizations, and (3) the failure of single Env vaccines to reproducibly elicit responses toward heterologous Env, regardless of the vaccination regimen utilized. Results encourage the use of poly-Env vaccine cocktails administered via DNA/VV/protein regimens in future non-human primate and clinical studies.

Antibody response to influenza infection of mice: different patterns for glycoprotein and nucleocapsid antigens

Respiratory syncytial virus (RSV) is a serious disease of children, responsible for an estimated 160,000 deaths per year worldwide. Despite the ongoing need for global prevention of RSV and decades of research, there remains no licensed vaccine. Sendai virus (SeV) is a mouse parainfluenza virus-type 1 which has been previously shown to confer protection against its human cousin, human parainfluenza virus-type 1 in African green monkeys (AGM). Here is described the study of a RSV vaccine (SeVRSV), produced by reverse genetics technology using SeV as a backbone to carry the full-length gene for RSV F. To test for immunogenicity, efficacy and safety, the vaccine was administered to AGM by intratracheal (i.t.) and intranasal (i.n.) routes. Control animals received the empty SeV vector or PBS. There were no booster immunizations. SeV and SeVRSV were cleared from the URT and LRT of vaccinated animals by day 10. Antibodies with specificities toward SeV and RSV were detected in SeVRSV primed animals as early as day ten after immunizations in both sera and nasal wash samples. One month after immunization all test and control AGM received an i.n. challenge with RSV-A2. SeVRSV-vaccinated animals exhibited reduced RSV in the URT compared to controls, and complete protection against RSV in the LRT. There were no clinically relevant adverse events associated with vaccination either before or after challenge. These data encourage advanced testing of the SeVRSV vaccine candidate in clinical trials for protection against RSV.

Human PIV-2 recombinant Sendai virus (rSeV) elicits durable immunity and combines with two additional rSeVs to protect against hPIV-1, hPIV-2, hPIV-3, and RSV.

Our previous studies of C57BL/6 mice intranasally infected with influenza virus (A/PR8) revealed a spike of virus‐specific immunoglobulin A (IgA)‐secreting antibody‐forming cells (AFC) in the mediastinal lymph node (MLN) 7 days post‐infection. Here we show that these AFC are directed only against viral glycoprotein, and not nucleocapsid antigens. The early IgA spike associates with a decline in glycoprotein‐specific AFC during week 2 post‐infection. In contrast to the glycoprotein‐specific AFC, nucleocapsid‐specific, IgA‐secreting AFC develop gradually in the MLN and persist for more than 3 weeks post‐infection. As peripheral lymph node reactions wane, the nucleocapsid‐specific AFC appear as long‐sustained populations in the bone marrow. Microanatomical examination of the respiratory tract in infected mice shows foci of infection established in the lung 2 days post‐infection, from which virus spreads to infect the entire lining of the trachea by day 3. At this time, viral haemagglutinin can be seen within the MLN, probably on projections from infected dendritic cells. This feature disappears within a day, though viral antigen expression continues to spread throughout the respiratory tract. Total IgA‐ and IgG‐secreting AFC appear histologically in large numbers during the first week post‐infection, significantly preceding the appearance of germinal centres (revealed by peanut agglutinin staining in week 2). To explain these results, we suggest that the initial immunogenic encounter of B cells with viral antigens occurs about 3 days post‐infection in the MLN, with antigens transported by dendritic cells from airway mucosa, the only site of viral replication. Viral glycoproteins expressed as integral membrane components on the surface of infected dendritic cells [probably in the absence of cognate T helper (Th) cells] promote members of expanding relevant B‐cell clones to undergo an IgA switch and terminal local plasmacytoid differentiation. Anti‐glycoprotein specificities are thus selectively depleted from progeny of activated B‐cell clones which are channelled to participate in germinal centre formation (perhaps by cognate T helper cells when they become sufficiently frequent). One product of the germinal centre reaction is the long‐sustained, bone marrow‐resident population, which is accordingly rich in anti‐nucleoprotein, but not anti‐glycoprotein specificities. Of note, we find that AFC responses toward influenza virus and Sendai virus differ, even though viral replication is limited to the airway mucosa in each case. The response towards Sendai virus exhibits neither the early appearance of anti‐glycoprotein AFC expressing IgA in draining lymph nodes, nor the subsequent relative deficit of this specificity from bone marrow AFC populations.

Respiratory Syncytial Virus: Current Progress in Vaccine Development

The human parainfluenza viruses (hPIVs) and respiratory syncytial viruses (RSVs) are the leading causes of hospitalizations due to respiratory viral disease in infants and young children, but no vaccines are yet available. Here we describe the use of recombinant Sendai viruses (rSeVs) as candidate vaccine vectors for these respiratory viruses in a cotton rat model. Two new Sendai virus (SeV)-based hPIV-2 vaccine constructs were generated by inserting the fusion (F) gene or the hemagglutinin-neuraminidase (HN) gene from hPIV-2 into the rSeV genome. The inoculation of either vaccine into cotton rats elicited neutralizing antibodies toward both homologous and heterologous hPIV-2 virus isolates. The vaccines elicited robust and durable antibodies toward hPIV-2, and cotton rats immunized with individual or mixed vaccines were fully protected against hPIV-2 infections of the lower respiratory tract. The immune responses toward a single inoculation with rSeV vaccines were long-lasting and cotton rats were protected against viral challenge for as long as 11 months after vaccination. One inoculation with a mixture of the hPIV-2-HN-expressing construct and two additional rSeVs (expressing the F protein of RSV and the HN protein of hPIV-3) resulted in protection against challenge viruses hPIV-1, hPIV-2, hPIV-3, and RSV. Results identify SeV vectors as promising vaccine candidates for four different paramyxoviruses, each responsible for serious respiratory infections in children.

Sendai virus recombinant vaccine expressing hPIV-3 HN or F elicits protective immunity and combines with a second recombinant to prevent hPIV-1, hPIV-3 and RSV infections

Respiratory syncytial virus (RSV) is the etiological agent for a serious lower respiratory tract disease responsible for close to 200,000 annual deaths worldwide. The first infection is generally most severe, while re-infections usually associate with a milder disease. This observation and the finding that re-infection risks are inversely associated with neutralizing antibody titers suggest that immune responses generated toward a first RSV exposure can significantly reduce morbidity and mortality throughout life. For more than half a century, researchers have endeavored to design a vaccine for RSV that can mimic or improve upon natural protective immunity without adverse events. The virus is herein described together with the hurdles that must be overcome to develop a vaccine and some current vaccine development approaches.