Xiaoyan Zhan

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

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.

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.

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

The human parainfluenza viruses (hPIVs) and respiratory syncytial virus (RSV) are the leading causes of serious respiratory illness in the human pediatric population. Despite decades of research, there are currently no licensed vaccines for either the hPIV or RSV pathogens. Here we describe the testing of hPIV-3 and RSV candidate vaccines using Sendai virus (SeV, murine PIV-1) as a vector. SeV was selected as the vaccine backbone, because it has been shown to elicit robust and durable immune activities in animal studies, and has already advanced to human safety trials as a xenogenic vaccine for hPIV-1. Two new SeV-based hPIV-3 vaccine candidates were first generated by inserting either the fusion (F) gene or hemagglutinin-neuraminidase (HN) gene from hPIV-3 into SeV. The resultant rSeV-hPIV3-F and rSeV-hPIV3-HN vaccines expressed their inserted hPIV-3 genes upon infection. The inoculation of either vaccine into cotton rats elicited binding and neutralizing antibody activities, as well as interferon-gamma-producing T cells. Vaccination of cotton rats resulted in protection against subsequent challenges with either homologous or heterologous hPIV-3. Furthermore, vaccination of cotton rats with a mixture of rSeV-hPIV3-HN and a previously described recombinant SeV expressing the F protein of RSV resulted in protection against three different challenge viruses: hPIV-3, hPIV-1 and RSV. Results encourage the continued development of the candidate recombinant SeV vaccines to combat serious respiratory infections of children.

Clustering of Th cell epitopes on exposed regions of HIV envelope despite defects in antibody activity

A long-standing question in the field of immunology concerns the factors that contribute to Th cell epitope immunodominance. For a number of viral membrane proteins, Th cell epitopes are localized to exposed protein surfaces, often overlapping with Ab binding sites. It has therefore been proposed that Abs on B cell surfaces selectively bind and protect exposed protein fragments during Ag processing, and that this interaction helps to shape the Th cell repertoire. While attractive in concept, this hypothesis has not been thoroughly tested. To test this hypothesis, we have compared Th cell peptide immunodominance in normal C57BL/6 mice with that in C57BL/6μMT/μMT mice (lacking normal B cell activity). Animals were first vaccinated with DNA constructs expressing one of three different HIV envelope proteins, after which the CD4+ T cell response profiles were characterized toward overlapping peptides using an IFN-γ ELISPOT assay. We found a striking similarity between the peptide response profiles in the two mouse strains. Profiles also matched those of previous experiments in which different envelope vaccination regimens were used. Our results clearly demonstrate that normal Ab activity is not required for the establishment or maintenance of Th peptide immunodominance in the HIV envelope response. To explain the clustering of Th cell epitopes, we propose that localization of peptide on exposed envelope surfaces facilitates proteolytic activity and preferential peptide shuttling through the Ag processing pathway.

Epstein-Barr virus vaccine development: a lytic and latent protein cocktail.

Epstein-Barr Virus (EBV) is the causative agent of acute infectious mononucleosis and associates with malignancies such as Burkitt lymphoma, nasopharyngeal carcinoma, and non-Hodgkins lymphoma. Additionally, EBV is responsible for B-lymphoproliferative disease in the context of HIV-infection, genetic immunodeficiencies and organ/stem-cell transplantation. Here we discuss past and current efforts to design an EBV vaccine. We further describe preliminary studies of a novel cocktail vaccine expressing both lytic and latent EBV proteins. Specifically, a tetrameric vaccinia virus (VV) -based vaccine was formulated to express the EBV lytic proteins gp350 and gp110, and the latent proteins EBNA-2 and EBNA-3C. In a proof-of-concept study, mice were vaccinated with the individual or mixed VV. Each of the passenger genes was expressed in vivo at levels sufficient to elicit binding antibody responses. Neutralizing gp350-specific antibodies were also elicited, as were EBV-specific T-cell responses, following inoculation of mice with the single or mixed VV. Results encourage further development of the cocktail vaccine strategy as a potentially powerful weapon against EBV infection and disease in humans.

Inhibition of ex vivo-expanded cytotoxic T-lymphocyte function by high-dose cyclosporine.

Background. Donor-derived, ex vivo-expanded cytotoxic T lymphocytes (CTL) can provide stem-cell transplantation (SCT) patients with a renewed capacity for virus-specific immune surveillance. Because SCT patients are often treated with cyclosporine (CsA), we questioned whether ex vivo-expanded CTL were susceptible to inhibition by this immunosuppressive drug. Methods. Human Epstein-Barr virus (EBV)-specific CTL were established by cultivating T cells for at least 5 weeks with interleukin (IL)-2 and irradiated, autologous EBV-transformed B-lymphoblastoid cell lines (LCL). In some cases, CsA was added during the last week of T-cell expansion. Effectors were then tested for cytotoxicity toward their targets in a chromium-release assay or by coculture with viable, unlabeled targets, in the presence or absence of CsA. Alloreactive CTL were similarly expanded and tested against major histocompatibility complex-mismatched stimulator cells. Results. CsA had a marginal effect on CTL function when added at concentrations greater than or equal to 250 ng/mL during the 4- to 6-hour chromium release assay. However, exposure of CTL to CsA for 1 week before assay reduced lytic function significantly. When the CTL lines were cocultured with viable targets in the presence of CsA, effectors were unable to eliminate their targets, which ultimately dominated the culture. Conclusions. We suggest that the activity of ex vivo-expanded CTL may be significantly compromised in the presence of high-dose CsA in vivo, particularly if CTL are administered for the purpose of long-term virus-specific immune surveillance.

HIV-1 vaccine development: tackling virus diversity with a multi-envelope cocktail.

A major obstacle to the design of a global HIV-1 vaccine is viral diversity. At present, data suggest that a vaccine comprising a single antigen will fail to generate broadly reactive B-cell and T-cell responses able to confer protection against the diverse isolates of HIV-1. While some B-cell and T-cell epitopes lie within the more conserved regions of HIV-1 proteins, many are localized to variable regions and differ from one virus to the next. Neutralizing B-cell responses may vary toward viruses with different i) antibody contact residues and/or ii) protein conformations while T-cell responses may vary toward viruses with different (i) T-cell receptor contact residues and/or (ii) amino acid sequences pertinent to antigen processing. Here we review previous and current strategies for HIV-1 vaccine development. We focus on studies at St. Jude Childrens Research Hospital (SJCRH) dedicated to the development of an HIV-1 vaccine cocktail strategy. The SJCRH multi-vectored, multi-envelope vaccine has now been shown to elicit HIV-1-specific B- and T-cell functions with a diversity and durability that may be required to prevent HIV-1 infections in humans.

HIV vaccines: brief review and discussion of future directions

A major barrier to the design of a successful HIV vaccine is virus diversity,which is particularly apparent in the envelope glycoprotein, the target of neutralizing antibodies. An antibody generated to one envelope glycoprotein may not recognize an isolate bearing a different envelope glycoprotein. Thus, single-envelope glycoprotein vaccines have protected against homologous but not necessarily against heterologous challenge. Antigenic diversity has been addressed in the design of vaccines for other pathogens by the preparation of polyvalent vaccines. The poliovirus vaccine, for example, comprises three serotypes of poliovirus, a feature that was essential in providing full protection against polio infection. Similarly, the authors propose that overcoming HIV diversity is likely to require the administration of a cocktail of envelope glycoprotein antigens. Delivery of such an array of envelope glycoproteins will elicit a broad immune response that is potentially capable of recognizing the diverse population of HIV-1 isolates. This article reviews data relevant to the development of cocktail vaccines which have been designed to elicit a wide range of envelope glycoprotein-specific B- and T-cell responses.

Limited Breadth of a T-Helper Cell Response to a Human Immunodeficiency Virus Envelope Protein

ABSTRACT Single-envelope human immunodeficiency virus (HIV) vaccines have been studied for more than a decade, with some successes in homologous challenge experiments in nonhuman primates but with no clear successes in clinical trials. To gain insight into the breadth of the immunity elicited by such vaccines, we have dissected the T-helper cell response of C57BL/6 mice to an individual, molecularly cloned envelope protein. Here, we report that T-helper cells responsive to HIV type 1 1035 envelope are very highly restricted in C57BL/6 animals: seven different hybridomas recovered from five separate mice recognized the same peptide, PKVSFEPIPIHYCAP, located in the C2 region of gp120. Three of these hybridomas were tested on a natural variant of the peptide but failed to respond. A more extensive analysis of whole splenic populations from other C57BL/6 mice immunized with the 1035 envelope reproducibly confirmed that the gp120-specific T-helper response was almost exclusively focused on a single epitope. We conclude that single-envelope vaccines may frequently fail to provoke an immune response sufficiently diverse to recognize variant sequences among circulating HIV. The results encourage the inclusion of more than one envelope in future vaccines to enhance the potential diversity and respective surveillance capacities of responding T-helper cell populations.

HIV vaccine rationale, design and testing.

A central obstacle to the design of a global HIV vaccine is viral diversity. Antigenic differences in envelope proteins result in distinct HIV serotypes, operationally defined such that antibodies raised against envelope molecules from one serotype will not bind envelope molecules from a different serotype. The existence of serotypes has presented a similar challenge to vaccine development against other pathogens. In such cases, antigenic diversity has been addressed by vaccine design. For example, the poliovirus vaccine includes three serotypes of poliovirus, and Pneumovax presents a cocktail of 23 pneumococcal variants to the immune system. It is likely that a successful vaccine for HIV must also comprise a cocktail of antigens. Here, data relevant to the development of cocktail vaccines, designed to harness diverse, envelope-specific B-cell and T-cell responses, are reviewed.