Live attenuated measles vaccine as a potential multivalent pediatric vaccination vector.

Live attenuated RNA viruses make highly efficient vaccines. Among them is the live attenuated measles virus (MV) vaccine that has been given to a very large number of children and has been shown to be highly efficacious and safe. MV vaccine induces a life-long immunity after a single injection or two low-dose injections. It is easily produced on a large scale in most countries and can be distributed at low cost. Reversion to pathogenicity has never been observed with this vaccine. For all of these characteristics, developing of MV vaccine vector as a multivalent vaccine to immunize children against both measles and other infectious agents such as human immunodeficiency virus (HIV), flaviviruses, or malaria might be very promising for worldwide use. As MV vaccine is inexpensive to produce, the generation of recombinant vaccines may remain affordable and attractive for the developing word. In this article, we describe the development of MV vector and present some recent data showing the capacity of recombinant MV vaccine to express various proteins from HIV and West Nile virus. In addition, the ability of recombinant MV to induce specific immune responses against these different pathogens are presented and discussed.

Measles virus.

Measles was an inevitable infection during the human development with substantial degree of morbidity and mortality. The severity of measles virus (MV) infection was largely contained by the development of a live attenuated vaccine that was introduced into the vaccination programs. However, all efforts to eradicate the disease failed and continued to annually result in significant deaths. The development of molecular biology techniques allowed the rescue of MV from cDNA that enabled important insights into a variety of aspects of the biology of the virus and its pathogenesis. Subsequently these technologies facilitated the development of novel vaccine candidates that induce immunity against measles and other pathogens. Based on the promising prospective, the use of MV as a recombinant vaccine and a therapeutic vector is addressed.

An immune competent mouse model for the characterization of recombinant measles vaccines.

Today, immune compromised interferon-α-receptor deficient mice expressing hCD46 (IFNARCD46tg) are usually used for measles virus (MV) based vaccine characterization. However, for the development of MV-based recombinant vaccine candidates (rMV), an immune competent mouse model is desirable in order to induce and evaluate meaningful immune response. In this study, humoral and cellular immune response induced by rMV in immune competent mice expressing human MV receptor CD46 (hCD46tg) were compared with those induced in wild-type black/6, and IFNARCD46tg mice.   All three strains developed humoral and cellular response against MV, whereas only hCD46tg and IFNARCD46tg mice developed a humoral response against the transgene. Differences were observed in the magnitude of the response, where the IFNARCD46tg mice displayed the strongest immune responses, followed by the hCD46tg mice and the black/6 mice. Interestingly, hCD46tg and wt black/6 mice showed a predominant CD4(+) T-cell response against MV-N, whereas IFNARCD46tg mice developed both, CD4(+) and CD8(+) T-cell response against MV-N. Analysis of the cytokine profile of MV-N specific CD4(+) T-cells and transgene (SIVgag) specific CD8(+) T-cells revealed qualitative differences of the T-cell responses; noticeably a significant reduction of the frequency of CD4(+)IL-2(+) expressing cells in IFNARCD46tg mice as compared with hCD46tg or wt black/6 mice. We show in this study significant quantitative and qualitative differences in immune responses between immune competent and immune-compromised mice. Our results therefore highlight the importance of the animal model and support the use of hCD46tg mice as mouse model for the characterization of the immunological profile induced by recombinant measles virus vaccine candidates.

Applications and challenges of multivalent recombinant vaccines.

The exceptional discoveries of antigen/gene delivery systems have allowed the development of novel prophylactic and therapeutic vaccine candidates. The vaccine candidates employ various antigen-delivery systems, particularly recombinant viral vectors. Recombinant viral vectors are experimental vaccines similar to DNA vaccines, but they use attenuated viruses or bacterium as a carrier "vector" to introduce microbial DNA to cells of the body. They closely mimic a natural infection and therefore can efficiently stimulate the immune system. Although such recombinant vectors may face extensive preclinical testing and will possibly have to meet stringent regulatory requirements, some of these vectors (e.g. measles virus vectors) may benefit from the profound industrial and clinical experience of the parent vaccine. Most notably, novel vaccines based on live attenuated viruses combine the induction of broad, strong and persistent immune responses with acceptable safety profiles. We assess certain technologies in light of their use against human immunodeficiency virus (HIV).

Relevance of a pre-existing measles immunity prior immunization with a recombinant measles virus vector.

Measles virus (MV) vectors are promising candidates for designing new recombinant vaccines since the parental live vaccines have a well-known safety and efficacy record. Like all viral vectors, the MV vector efficacy in inducing a protecting immune answer could be affected by the pre-existing immunity among the human population. In order to determine the optimal immunization route and regimen, we mimicked a MV pre-immunity by passively administrating MV neutralizing antibodies (MV-nAb) prior intramuscular (i.m.) and/or intranasal (i.n.) immunization with recombinant MV expressing the SIV-gag antigen (rMV-SIVgag). Our results revealed that 500 mIU of MV-nAb allowed the induction of a humoral and cellular immune response against the vector and the transgene, while higher titers of the MV-nAb were significantly inhibitory. In a prime-boost regimen, in the presence of MV-nAb, the intranasal-intramuscular (i.n.-i.m.) or intramuscular-intramuscular (i.m.-i.m.) routes induced higher humoral immune responses against the vector and the transgene (SIV-gag). In naive animals, cellular immune response was significantly higher by i.m. immunization; however, MV pre-immunity did not seem to affect the cellular immune response after an i.n. immunization.   In summary, we show that a pre-existing immunity of up to 500 mIU anti-MV neutralizing antibodies had little effect on the replication of rMV and did not inhibit the induction of significant humoral and cellular immune responses in immune-competent mice.

Sequence and immunogenicity of a clinically approved novel measles virus vaccine vector.

The measles virus vaccine (MVbv) is a clinically certified and well-tolerated vaccine strain that has been given both parenterally and mucosally. It has been extensively used in children and has proven to be safe and effective in eliciting protective immunity. This specific strain was therefore chosen to generate a measles viral vector. The genome of the commercial MVbv vaccine strain was isolated, sequenced and a plasmid, p(+)MVb, enabling transcription of the viral antigenome and rescue of MVb, was constructed. Phylogenic and phenotypic analysis revealed that MVbv and the rescued MVb constitute another evolutionary branch within the hitherto classified measles vaccines. Plasmid p(+)MVb was modified by insertion of artificial MV-type transcription units (ATUs) for the generation of recombinant viruses (rMVb) expressing additional proteins. Replication characteristics and immunogenicity of rMVb vectors were similar to the parental MVbv and to other vaccine strains. The expression of the additional proteins was stable over 10 serial virus transfers, which corresponds to an amplification greater than 10 ( 20) . The excellent safety record and its efficient application as aerosol may add to the usefulness of the derived vectors.

Priming T-cell responses with recombinant measles vaccine vector in a heterologous prime-boost setting in non-human primates.

Licensed live attenuated virus vaccines capable of expressing transgenes from other pathogens have the potential to reduce the number of childhood immunizations by eliciting robust immunity to multiple pathogens simultaneously. Recombinant attenuated measles virus (rMV) derived from the Edmonston Zagreb vaccine strain was engineered to express simian immunodeficiency virus (SIV) Gag protein for the purpose of evaluating the immunogenicity of rMV as a vaccine vector in rhesus macaques. rMV-Gag immunization alone elicited robust measles-specific humoral and cellular responses, but failed to elicit transgene (Gag)-specific immune responses, following aerosol or intratracheal/intramuscular delivery. However, when administered as a priming vaccine to a heterologous boost with recombinant adenovirus serotype 5 expressing the same transgene, rMV-Gag significantly enhanced Gag-specific T lymphocyte responses following rAd5 immunization. Gag-specific humoral responses were not enhanced, however, which may be due to either the transgene or the vector. Cellular response priming by rMV against the transgene was highly effective even when using a suboptimal dose of rAd5 for the boost. These data demonstrate feasibility of using rMV as a priming component of heterologous prime-boost vaccine regimens for pathogens requiring strong cellular responses.

Recombinant measles virus-HPV vaccine candidates for prevention of cervical carcinoma.

Cervical cancer is mainly associated with HPV genotype 16 infection. Recombinant measles virus (rMV) expressing HPV genotype 16 L1 capsid protein was generated by construction of an antigenomic plasmid, followed by rescue using the human "helper" cell line 293-3-46. In cell cultures the recombinant MV-L1 virus replicated practically as efficiently as the standard attenuated MV established as commercial vaccine, devoid of the transgene. The high genetic stability of MVb2-L1 was confirmed by 10 serial viral transfers in cell culture. In transgenic mice expressing the MV receptor CD46 the recombinant induced strong humoral immune responses against both MV and HPV; the antibodies against L1 exhibited mainly neutralizing capacity. Our data suggest that MV is a promising vehicle for development of inexpensive and efficient vaccines protecting from HPV infection.

Recombinant measles viruses expressing single or multiple antigens of human immunodeficiency virus (HIV-1) induce cellular and humoral immune responses.

Recombinant measles viruses (rMV) based on the live attenuated measles vaccine strain (MVb) expressing antigens of HIV-1 clade B were generated by reverse genetics. Recombinants expressing single or double antigens of HIV-1 (rMV-HIV) were genetically highly stable on human diploid cells. The production process of these viruses was essentially similar to the parental MV strain, yielding comparative end titers. Immunization of tg-mice by different regimens and formulations showed potent humoral and cellular immune responses against MV and HIV antigens. Recombinant MV-HIV expressing Gag protein conferred protective immunity in tg-mice after a high-dose pseudochallenge with recombinant vaccinia virus. In addition, rMV-HIV boosted anti-HIV antibodies, in the presence of pre-existing anti-vector antibodies.

Induction of neutralising antibodies and cellular immune responses against SARS coronavirus by recombinant measles viruses.

Live attenuated recombinant measles viruses (rMV) expressing a codon-optimised spike glycoprotein (S) or nucleocapsid protein (N) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) were generated (rMV-S and rMV-N). Both recombinant viruses stably expressed the corresponding SARS-CoV proteins, grew to similar end titres as the parental strain and induced high antibody titres against MV and the vectored SARS-CoV antigens (S and N) in transgenic mice susceptible to measles infection. The antibodies induced by rMV-S had a high neutralising effect on SARS-CoV as well as on MV. Moreover, significant N-specific cellular immune responses were measured by IFN-gamma ELISPOT assays. The pre-existence of anti-MV antibodies induced by the initial immunisation dose did not inhibit boost of anti-S and anti-N antibodies. Immunisations comprising a mixture of rMV-S and rMV-N induced immune responses similar in magnitude to that of vaccine components administered separately. These data support the suitability of MV as a bivalent candidate vaccine vector against MV and emerging viruses such as SARS-CoV.

Use of viral vectors for the development of vaccines.

The exceptional discoveries of antigen/gene delivery systems have allowed the development of novel prophylactic and therapeutic vaccine candidates. This review highlights various antigen-delivery systems, particularly viral vectors, and assesses the underlying technologies in light of their use against AIDS and malaria. Although such recombinant vectors may face extensive preclinical testing and will possibly have to meet stringent regulatory requirements, some of these vectors may benefit from the profound industrial and clinical experience of the parent vaccine. Most notably, novel vaccines based on live, recombinant vectors may combine the induction of broad, strong and persistent immune responses with acceptable safety profiles.

Attenuated measles virus as a vaccine vector.

Live attenuated measles virus (MV) vaccines have an impressive record of safety, efficacy and ability to induce life-long immunity against measles infection. Using reverse genetics technology, such negative-strand RNA viruses can now be rescued from cloned DNA. This technology allows the insertion of exogenous genes encoding foreign antigens into the MV genome in such a way that they can be expressed by the MV vaccine strain, without affecting virus structure, propagation and cell targeting. Recombinant viruses rescued from cloned cDNA induce immune responses against both measles virus and the cloned antigens. The tolerability of MV to gene(s) insertion makes it an attractive flexible vector system, especially if broad immune responses are required. The fact that measles replication strictly occurs in the cytoplasm of infected cells without DNA intermediate has important biosafety implications and adds to the attractiveness of MV as a vector. In this article we report the characteristics of reporter gene expression (GFP, LacZ and CAT) and the biochemical, biophysical and immunological properties of recombinant MV expressing heterologous antigens of simian immunogeficiency virus (SIV).

Viral vectors for malaria vaccine development.

A workshop on viral vectors for malaria vaccine development, organized by the PATH Malaria Vaccine Initiative, was held in Bethesda, MD on October 20, 2005. Recent advancements in viral-vectored malaria vaccine development and emerging vector technologies were presented and discussed. Classic viral vectors such as poxvirus, adenovirus and alphavirus vectors have been successfully used to deliver malaria antigens. Some of the vaccine candidates have demonstrated their potential in inducing malaria-specific immunity in animal models and human trials. In addition, emerging viral-vector technologies, such as measles virus (MV), vesicular stomatitis virus (VSV) and yellow fever (YF) virus, may also be useful for malaria vaccine development. Studies in animal models suggest that each viral vector is unique in its ability to induce humoral and/or cellular immune responses. Those studies have also revealed that optimization of Plasmodium genes for mammalian expression is an important aspect of vaccine design. Codon-optimization, surface-trafficking, de-glycosylation and removal of toxic domains can lead to improved immunogenicity. Understanding the vector's ability to induce an immune response and the expression of malaria antigens in mammalian cells will be critical in designing the next generation of viral-vectored malaria vaccines.

Recombinant measles virus induces cytolysis of cutaneous T-cell lymphoma in vitro and in vivo.

Measles virus (MV) has shown promise as an oncolytic virus in the treatment of different tumor models for human B-cell lymphoma, multiple myeloma, ovarian cancer, and glioma. We have shown that, in a phase I clinical trial, MV vaccine induces tumor regression in cutaneous T-cell lymphoma (CTCL) patients. Here, we investigated in detail, the effect of recombinant MV (rMV) vaccine strain in CTCL cell cultures, and in vivo in established CTCL xenografts in nude mice. The susceptibility of three CTCL cell lines, originating from patients, to rMV was tested by determination of cell surface expression of MV receptors. All cell lines expressed the receptors CD150 and CD46 and were easily infected by rMV and induced complete cell lysis. The cytoreductive activity was apparent in cells forming aggregates, indicating a cell-to-cell spread of MV and cytolysis owing to virus infection. Intratumoral (i.t.) injection of rMV, expressing enhanced green fluorescent protein induced complete regression of large established human CTCL tumors in nude mice, whereas tumors with control treatment progressed exponentially. Immunohistochemical analysis of tumor biopsies, after i.t. treatment, for MV-NP protein complex demonstrated replication of MV within the tumors. The data demonstrate the potential of MV as a therapeutic agent against CTCL.

Oncolytic measles virus in cutaneous T-cell lymphomas mounts antitumor immune responses in vivo and targets interferon-resistant tumor cells.

Some cutaneous T-cell lymphomas, (CTCLs) clonal T cells are deficient in interferon signaling, making them promising targets for viral oncolysis. We evaluated cytopathic effects of measles virus (MV) in CTCL. CTCL cell lines and infiltrating lymphocytes in CTCL expressed MV receptors CD150 and CD46. In a phase 1 dose escalation trial a total of 16 injections of live MV, Edmonston-Zagreb vaccine strain, were given intratumorally to 5 patients with CTCL. Patients had antimeasles-serum antibodies and were pretreated with interferon-alpha to prevent uncontrolled virus spread. The well-tolerated treatment with MV resulted in clinical responses. Evaluation of biopsies, before and at 11 days after injection, by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated local viral activity with positive staining for MV nucleoprotein (NP), an increase of the interferon gamma (IFN-gamma)/CD4 and IFN-gamma/CD8 mRNA ratios and a reduced CD4/CD8 ratio. All patients demonstrated an increased antimeasles antibody titer after therapy. The data demonstrate that CTCLs are promising targets for an MV-based oncolytic therapy.

Measles virus spreads in rat hippocampal neurons by cell-to-cell contact and in a polarized fashion.

Measles virus (MV) can infect the central nervous system and, in rare cases, causes subacute sclerosing panencephalitis, characterized by a progressive degeneration of neurons. The route of MV transmission in neurons was investigated in cultured rat hippocampal slices by using MV expressing green fluorescent protein. MV infected hippocampal neurons and spread unidirectionally, in a retrograde manner, from CA1 to CA3 pyramidal cells and from there to the dentate gyrus. Spreading of infection depended on cell-to-cell contact and occurred without any detectable release of infectious particles. The role of the viral proteins in the retrograde MV transmission was determined by investigating their sorting in infected pyramidal cells. MV glycoproteins, the fusion protein (F) and hemagglutinin (H), the matrix protein (M), and the phosphoprotein (P), which is part of the viral ribonucleoprotein complex, were all sorted to the dendrites. While M, P, and H proteins remained more intracellular, the F protein localized to prominent, spine-type domains at the surface of infected cells. The detected localization of MV proteins suggests that local microfusion events may be mediated by the F protein at sites of synaptic contacts and is consistent with a mechanism of retrograde transmission of MV infection.

Measles virus and canine distemper virus target proteins into a TAP-independent MHC class I-restricted antigen-processing pathway.

After infection of CEM174.T2 cells [deficient for the transporter of antigen presentation (TAP)] with measles virus (MV) the nucleocapsid protein is recognized by L(d)-restricted cytotoxic T cells in a TAP-independent, chloroquine-sensitive fashion. Presentation via the TAP-independent pathway requires virus replication. During MV infection of the cell the nucleocapsid as well as the matrix protein enter the endolysosomal compartment as indicated by colocalization with the lysosomal-associated membrane protein 1 (LAMP-1). Similarly, the nucleocapsid protein of canine distemper virus (CDV) is recognized in a TAP-independent fashion. In addition, a recombinant MV expressing bacterial beta-galactosidase protein is able to introduce the recombinant antigen into the TAP-independent pathway whereas a vaccinia virus expressing beta-galactosidase is not. These data and a report about TAP-independent recognition of parainfluenza virus type 1 suggest that members of the Paramyxoviridae family regularly introduce viral proteins into the TAP-independent antigen-processing pathway.