# Patent

## Title
Recombinant measles viruses expressing epitopes of antigens of RNA viruses—use for the preparation of vaccine compositions

## Abstract

The invention relates to a recombinant measles virus expressing a heterologous amino acid sequence derived from an antigen of a determined RNA virus, the recombinant measles virus being capable of eliciting a humoral and/or cellular immune response against measles virus or against the RNA virus or against both measles virus and against the RNA virus. It also relates to the use of the recombinant measles virus for the preparation of immunogenic compositions.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This is a continuation of application Ser. No. 11/014,842, filed Dec. 20, 2004, now abandoned which is a continuation of PCT International Application No. PCT/EP2003/007146, filed Jun. 20, 2003, which claims the benefit of European Patent Application No. EP02291550.8, filed Jun. 20, 2002, all of which are incorporated herein by reference.
Sequence Listing:
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 8, 2012, is named DI200221.txt and is 122,056 bytes in size.


The invention relates to recombinant measles viruses expressing epitopes of antigens of RNA viruses including especially retroviruses and flaviviruses and to their use for the preparation of vaccine compositions.
Measles virus is a member of the order mononegavirales, i.e., viruses with a non-segmented negative-strand RNA genome. The non segmented genome of measles virus (MV) has an antimessage polarity which results in a genomic RNA which is not translated either in vivo or in vitro nor infectious when purified.
Transcription and replication of non-segmented (−) strand RNA viruses and their assembly as virus particles have been studied and reported especially in Fields virology (3rdedition, vol. 1, 1996, Lippincott—Raven publishers—Fields B N et al). Transcription and replication of measles virus do not involve the nucleus of the infected cells but rather take place in the cytoplasm of said infected cells. The genome of the measles virus comprises genes encoding six major structural proteins from the six genes (designated N, P, M, F, H and L) and an additional two-non structural proteins from the P gene. The gene order is the following: 3′-I, N, P (including C and V), M, F, H, and L large polymerase protein at the 5′ end. The genome further comprises non coding regions in the intergenic region M/F; this non-coding region contains approximately 1000 nucleotides of untranslated RNA. The cited genes respectively encode the leader peptide (I gene), the proteins of the nucleocapsid of the virus, i.e., the nucleoprotein (N), the phosphoprotein (P), and the large protein (L) which assemble around the genome RNA to provide the nucleocapsid. The other genes encode the proteins of the viral envelope including the hemagglutinin (H), the fusion (F) and the matrix (M) proteins.
The measles virus has been isolated and live attenuated vaccines have been derived from the Edmonston MV isolated in 1954 (Enders, J. F., and T. C. Peebles. 1954. Propagation in tissue cultures od cytopathogenic agents from patients with measles. Proc. Soc. Exp. Biol. Med. 86:277-286.), by serial passages on primary human kidney or amnion cells. The used strains were then adapted to chick embryo fibroblasts (CEF) to produce Edmonston A and B seeds (Griffin, D., and W. Bellini. 1996. Measles virus, p. 1267-1312. In B. Fields, D. Knipe, et al. (ed.), Virology, vol. 2. Lippincott—Raven Publishers, Philadelphia). Edmonston B was licensed in 1963 as the first MV vaccine. Further passages of Edmonston A and B on CEF produced the more attenuated Schwarz and Moraten viruses (Griffin, D., and W. Bellini. 1996. Measles virus, p. 1267-1312. In B. Fields, D. Knipe, et al. (ed.), Virology, vol. 2. Lippincott—Raven Publishers, Philadelphia) whose sequences have recently been shown to be identical (Parks, C. L., R. A. Lerch, P. Walpita, H. P. Wang, M. S. Sidhu, and S. A. Udem. 2001. Analysis of the noncoding regions of measles virus strains in the Edmonston vaccine lineage. J. Virol. 75:921-933; Parks, C. L., R. A. Lerch, P. Walpita, H. P. Wang, M. S. Sidhu, and S. A. Udem. 2001. Comparison of predicted amino acid sequences of measles virus strains in the Edmonston vaccine lineage. J. Virol. 75:910-920). Because Edmonston B vaccine was reactogenic, it was abandoned in 1975 and replaced by the Schwarz/Moraten vaccine which is currently the most widely used measles vaccine in the world (Hilleman, M. 2002. Current overview of the pathogenesis and prophylaxis of measles with focus on practical implications. Vaccine. 20:651-665). Several other vaccine strains are also used: AIK-C, Schwarz F88, CAM70, TD97 in Japan, Leningrad-16 in Russia, and Edmonston Zagreb. The CAM70 and TD97 Chinese strains were not derived from Edmonston. Schwarz/Moraten and AIK-C vaccines are produced on CEF. Zagreg vaccine is produced on human diploid cells (WI-38).
The live attenuated vaccine derived from the Schwarz strain is commercialized by Aventis Pasteur (Lyon France) under the trademark ROUVAX®.
In a noteworthy and pioneer work, Martin Billeter and colleagues cloned an infectious cDNA corresponding to the antigenome of Edmonston MV and established an original and efficient reverse genetics procedure to rescue the corresponding virus (Radecke, F., P. Spielhofer, H. Schneider, K. Kaelin, M. Huber, K. Dotsch, G. Christiansen, and M. Billeter., 1995. Rescue of measles viruses from cloned DNA. EMBO Journal. 14:5773-5784) and WO 97/06270. They developed an Edmonston vector for the expression of foreign genes (Radecke, F., and M. Billeter. 1997. Reverse genetics meets the nonsegmented negative-strand RNA viruses. Reviews in Medical Virology. 7:49-63.) and demonstrated its large capacity of insertion (as much as 5 kb) and its high stability at expressing transgenes (Singh, M., and M. Billeter. 1999. A recombinant measles virus expressing biologically active human interieukin-12. J. Gen. Virol. 80:101-106; Singh, M., R. Cattaneo, and M. Billeter. 1999. A recombinant measles virus expressing hepatitis B virus surface antigen induces humoral immune responses in genetically modified mice. J. Virol. 73:4823-4828; Spielhofer, P., T. Bachi, T. Fehr, G. Christiansen, R. Cattaneo, K. Kaelin, M. Billeter, and H. Nairn. 1998. Chimeric measles viruses with a foreign envelope. J. Virol. 72:2150-2159); Wang, Z., T. Hangartner, L. Comu, A. Martin, M. Zuniga, M. Billeter, and H. Nairn. 2001. Recombinant measles viruses expressing heterologous antigens of mumps and simian immunodeficiency viruses. Vaccine. 19:2329-2336. This vector was cloned from the Edmonston B strain of MV propagated in HeLa cells (Ballart, I., D. Eschle, R. Cattaneo, A. Schmid, M. Metzler, J. Chan, S. Pifko-Hirst, S. A. Udem, and M. A. Billeter. 1990. Infectious measles virus from cloned cDNA. Embo J. 9:379-384).
In addition, recombinant measles virus expressing Hepatitis B virus surface antigen has been produced and shown to induce humoral immune responses in genetically modified mice (Singh M. R. et al, 1999, J. virol. 73: 4823-4828).
MV vaccine induces a very efficient, life-long immunity after a single low-dose injection (104 TCID50) (33,34). Protection is mediated both by antibodies and by CD4+ and CD8+ T cells. The MV genome is very stable and reversion to pathogenicity has never been observed with this vaccine. MV replicates exclusively in the cytoplasm, ruling out the possibility of integration in host DNA. Furthermore, an infectious cDNA clone corresponding to the anti-genome of the Edmonston strain of MV and a procedure to rescue the corresponding virus have been established (35). This cDNA has been made into a vector to express foreign genes (36). It can accommodate up to 5 kb of foreign DNA and is genetically very stable (37, 38, 39).
From the observation that the properties of the measles virus and especially its ability to elicit high titers of neutralizing antibodies in vivo and its property to be a potent inducer of long lasting cellular immune response, the inventors have proposed that it may be a good candidate for the preparation of compositions comprising recombinant infectious viruses expressing antigenic peptides or polypeptides of determined RNA viruses, including especially retroviruses or flaviviruses, to induce neutralizing antibodies against said RNA virus and especially said retroviruses or flaviviruses which preferably could be suitable to achieve at least some degree of protection against said RNA viruses, especially retroviruses or flaviviruses, in animals and more preferably in human hosts. Especially, MV strains and in particular vaccine strains have been elected in the present invention as candidate vectors to induce immunity against both measles virus and RNA virus whose constituent is expressed in the designed recombinant MV, in exposed infant populations because they are having no MV immunity. Adult populations, even already MV immunized individuals, may however also benefit from MV recombinant immunization because re-administering MV virus under the recombinant form of the present invention may result in a boost of anti-MV antibodies.
Among retroviruses of interest, the inventors have chosen AIDS retroviruses, including HIV-1 and among flaviviruses, some which are important human pathogens such as Yellow Fever Virus (YFV) and West Nile Virus (WNV).
The YFV and WNV belong to the family Flaviviridae described in Fields virology (3rdedition, vol. 1, 1996, Lippincott—Raven publishers—Fields B N et al).
The invention relates to a recombinant mononegavirales virus expressing a heterologous amino acid sequence, said recombinant virus being capable of eliciting a humoral and/or a cellular immune response against said heterologous amino acid sequence including in individuals having pre-existing measles virus immunity.
In a first embodiment, the invention especially provides recombinant measles viruses capable of expressing antigens and especially epitopes derived from antigens of RNA viruses including retroviruses or flaviviruses.
The invention also relates to nucleic acid constructs especially to recombinant nucleic acid constructs expressing the recombinant measles viruses and expressing therewith antigens or epitopes of antigens of retroviruses or flaviviruses.
The invention concerns also processes for the preparation of such recombinant measles viruses and especially relates to the production of such recombinant MV in rescue systems.
The invention is also directed to compositions comprising said recombinant measles viruses as active principles for protection of hosts, especially human hosts, against diseases related to infections by said retroviruses, especially by AIDS retroviruses, or said flaviviruses, especially Yellow Fever Virus or West Nile Virus.
Nucleic acid sequences of Measles Viruses have been disclosed in International Patent Application WO 98/13501, especially a DNA sequence of 15,894 nucleotides corresponding to a DNA copy of the positive strand (antigenomic) message sense RNA of various wild-type of vaccine measles strains, including Edmonston Wild-type strain, Moraten strain and Schwarz strain which is identical to the Moraten strain except for nucleotide positions 4917 and 4924 where Schwarz strain has a <<C>> instead of a <<T>>.
In order to produce recombinant measles viruses, a rescue system has been developed for the Edmonston MV strain and described in International Patent Application WO 97/06270. The description of said rescue system contained in WO 97/06270 is incorporated herewith by reference, and reference is made especially to the examples of this International application, including the Examples related to cells and viruses, to generation of cell line 293-3-46, plasmid constructions, transfection of plasmids and harvest of reporter gene products, experimental set-up to rescue MV, helper cells stably expressing MV N and P proteins as well as T7 RNA polymerase, MV rescue using helper cells 293-3-46 and characterization of rescued MV.
The rescue system disclosed in WO 97/06270 has been further developed to include a heat-shock step described in Parks C. L. et al, 1999, J. virol. 73: 3560-3566. The disclosure of this enhanced measles virus cDNA rescue system is incorporated herewith by reference.
The invention thus relates to recombinant measles viruses expressing a heterologous amino acid sequence derived from an antigen of a determined RNA virus, especially from a retrovirus or flavivirus, wherein said recombinant measles virus is capable of eliciting a humoral and/or a cellular immune response against measles virus or against said RNA virus, especially retrovirus or flavivirus or against both measles virus and against said RNA virus, especially retrovirus or flavivirus.
The expression <<heterologous amino acid sequence>> is directed to an amino acid sequence which is not derived from the antigens of measles viruses, said heterologous amino acid sequence being accordingly derived from a RNA virus, especially from a retrovirus or flavivirus of interest in order to establish an immune response in a host, especially in a human and preferably to establish protection against an infection by said RNA virus, especially retrovirus or flavivirus.
The heterologous amino acid sequence expressed in recombinant measles viruses according to the invention is such that it is capable of eliciting a humoral and/or cellular immune response in a determined host, against the RNA virus, especially retrovirus or flavivirus from which it originates. Accordingly, this amino acid sequence is one which comprises at least one epitope of an antigen, especially a conserved epitope, which epitope is exposed naturally on the antigen or is obtained or exposed as a result of a mutation or modification or combination of antigens.
Antigens used for the preparation of the recombinant measles viruses are especially envelope antigens of RNA viruses such as retroviruses or flaviviruses, especially from envelopes of AIDS viruses including HIV-1 or from envelopes of the Yellow Fever Virus or envelopes from the West Nile Virus. Other retroviral or flaviviral antigens may however be advantageously used in order to derive recombinant measles viruses capable of eliciting antibodies against said retroviruses or flaviviruses, and the invention relates in a particular embodiment to antigens from which amino acid sequences can be derived which elicit the production of neutralizing antibodies against the retrovirus or flavivirus. According to another embodiment of the invention, amino acid sequence of these antigens alternatively or additionally also elicits a cellular immune response against the retrovirus or flaviviruses.
Advantageously, the recombinant measles virus of the invention also elicits a humoral and/or cellular immune response against measles virus. This response is however not mandatory provided the immune response against the RNA virus, especially retrovirus or flavivirus is indeed obtained.
According to a preferred embodiment of the invention, the recombinant measles virus of the invention is obtained within a rescue system for the preparation of infectious measles viruses. Accordingly, the recombinant measles virus is a rescued infectious measles virus recovered from a rescue system.
A particular recombinant measles virus of the invention is derived from the Edmonston strain of measles virus.
Another particular and preferred recombinant measles virus according to the invention is derived from the Schwarz strain and especially from an approved vaccine Schwarz strain such as that produced under the trademark ROUVAX®, available from Aventis Pasteur (France).
The invention thus provides for a recombinant measles virus which is recovered from helper cells transfected with a cDNA encoding the antigenomic RNA ((+)strand) of the measles virus, said cDNA being recombined with a nucleotide sequence encoding the RNA viral, especially retroviral or flaviviral, heterologous amino acid sequence.
The expression <<encoding>> in the above definition encompasses the capacity of the cDNA to allow transcription of a full length antigenomic (+)RNA, said cDNA serving especially as template for transcription. Accordingly, when the cDNA is a double stranded molecule, one of the strands has the same nucleotide sequence as the antigenomic (+) strand RNA of the measles virus, except that <<U>> nucleotides are substituted by <<T>> in the cDNA. Such a cDNA is for example the insert corresponding to the measles virus, contained in the pTM-MVSchw plasmid deposited under No I-2889 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES (CNCM), 25 rue du Docteur Roux, F-75724 Paris Cedex 15, France on Jun. 12, 2002. This plasmid is represented on FIG. 2A.
The expression “cDNA” used for the description of the nucleotide sequence of the molecule of the invention merely relates to the fact that originally said molecule is obtained by reverse transcription of the full length genomic (−)RNA genome of viral particles of the measles virus.
This should not be regarded as a limitation for the methods used for its preparation. The invention thus encompasses, within the expression “cDNA”, every DNA provided it has the above defined nucleotide sequence. Purified nucleic acids, including DNA are thus encompassed within the meaning cDNA according to the invention, provided said nucleic acid, especially DNA fulfils the above-given definitions.
The helper cells according to the rescue system are transfected with a transcription vector comprising the cDNA encoding the full length antigenomic (+)RNA of the measles virus, when said cDNA has been recombined with a nucleotide sequence encoding the heterologous amino acid sequence of interest (heterologous nucleotide sequence) and said helper cells are further transfected with an expression vector or several expression vectors providing the helper functions including those enabling expression of trans-acting proteins of measles virus, i.e., N, P and L proteins and providing expression of an RNA polymerase to enable transcription of the recombinant cDNA and replication of the corresponding viral RNA.
The invention relates in particular to the preparation of recombinant measles viruses bearing epitopes of antigens of HIV retroviruses. It encompasses especially a recombinant measles virus expressing a heterologous amino acid sequence which is derived from an envelope antigen of HIV and which is especially derived from an envelope protein or glycoprotein of HIV-1.
The antigens of interest in this respect are especially gp160, gp120 and gp41 of HIV-1 or gp140, GAG or TAT of HIV-1.
In a particular embodiment of the invention, the heterologous amino acid sequence is derived from a recombinant gp160, gp120 of HIV-1 or gp140, GAG or TAT of HIV-1.
The invention is directed in particular to a recombinant measles virus wherein the V1, V2 and/or V3 loops of the gp120 (or gp160) antigen are deleted or deleted in part, individually or in combination in such a way that conserved epitopes are exposed on the obtained recombinant gp120 antigen.
The V1, V2 and V3 loops of the gp120 (or gp160) antigen of HIV-1 have been especially disclosed in Fields virology (Fields B. N. et al—Lippincott Raven publishers 1996, p. 1953-1977).
According to another embodiment of the invention, the recombinant measles virus is such that it expresses a heterologous amino acid sequence derived from the gp120 (or gp160) antigen of HIV-1, wherein the V1, V2 and/or V3 loops of the gp120 (or gp160) antigen are substituted or substituted in part, individually or in combination, in such a way that conserved epitopes are exposed on the obtained recombinant gp120 (or gp160) antigen.
According to another particular embodiment, the recombinant measles virus expressing a heterologous DNA sequence derived from an envelope antigen of HIV-1 is derived from the gp120 antigen in such a way that the V1 and V2 loops are deleted and the V3 loop is substituted for the sequence AAELDKWASAA (SEQ ID NO: 8).
According to another particular embodiment of the invention, the recombinant measles virus is one expressing an heterologous amino acid sequence selected among gp160ΔV3, gp160ΔV1V2, gp160ΔV1V2V3, gp140ΔV3, gp140ΔV1V2, gp140ΔV1V2V3, which heterologous amino acid sequences are schematically represented on FIG. 1.
The invention also relates to recombinant measles viruses as defined according to the above statements, wherein the amino acid sequence is derived from an antigen of the Yellow Fever virus selected among the envelope (Env) or the NS1 proteins or immunogenic mutants thereof.
The invention also relates to recombinant measles viruses as defined according to the above statements, wherein the amino acid sequence is derived from an antigen of the West Nile virus selected among the envelope (E), premembrane (preM) or immunogenic mutants thereof.
The invention also relates to recombinant measles viruses or to virus like particles (VLP) which express double or multiple recombinant antigens, especially multiple HIV antigens (including fragments thereof) or flavivirus antigens, against which an immune response is sought. Such recombinant measles viruses or VLP may advantageously express antigens from different viruses and thus provide immunogens against various viruses.
The invention further relates to recombinant measles viruses according to anyone of the above definitions, wherein the cDNA required for the expression of the viral particles, which is comprised within the EdB-tag virus vector or preferably within the pTM-MVSchw vector is recombined with the ATU sequence of FIG. 8, said ATU being inserted in a position of the EdB-tag vector or of the pTM-MVSchw vector taking advantage of the gradient of the viral genome to allow various levels of expression of the transgenic sequence encoding the heterologous amino acid sequence inserted in said ATU. The invention advantageously enables the insertion of such heterologous DNA sequences in a sequence which is designated an Additional Transcription Unit (ATU) especially an ATU as disclosed by Billeter et al in WO 97/06270.
The advantageous immunological properties of the recombinant measles viruses according to the invention can be shown in an animal model which is chosen among animals susceptible to measles viruses and wherein the humoral and/or cellular immune response against the heterologous antigen and/or against the measles virus is determined.
Among such animals suitable to be used as model for the characterization of the immune response, the skilled person can especially use mice and especially recombinant mice susceptible to measles viruses, or in monkeys.
In a preferred embodiment of the invention, the recombinant measles virus of the invention is suitable to elicit neutralizing antibodies against the heterologous amino acid sequence in a mammalian animal model susceptible to measles virus. Especially, this immune response comprising elicitation of neutralizing antibodies can be sought in recombinant mice or monkeys.
According to another particular embodiment of the invention, especially when the heterologous amino acid sequence is derived from one of the envelope proteins of HIV-1 and where it elicits antibodies capable of neutralizing a primary HIV isolate, the response is advantageously tested on indicator cells such as P4-CCR5 cells available from the NIH (NIH AIDS Research and Reference Reagent Program). (Charneau P. et al—1994—J. Mol. Biol. 241: 651-662).
According to another preferred embodiment, the recombinant measles virus according to the invention elicits neutralizing antibodies against the heterologous amino acid sequence in a mammal, with a titre of at least 1/40000 when measured in ELISA, and a neutralizing titre of at least 1/20.
The invention also relates to a recombinant measles virus nucleotide sequence comprising a replicon comprising (i) a cDNA sequence encoding the full length antigenomic (+)RNA of measles virus operatively linked to (ii) an expression control sequence and (iii) a heterologous DNA sequence coding for a determined heterologous amino acid sequence, said heterologous DNA sequence being cloned in said replicon in conditions allowing its expression and in conditions not interfering with transcription and replication of said cDNA sequence, said replicon having a total number of nucleotides which is a multiple of six.
A particular cDNA sequence is the sequence of the cDNA of the Schwarz strain depicted on FIG. 11. Such a cDNA can be obtained from pTM-MVSchw.
pTM-MVSchw is a plasmid derived from Bluescript containing the complete sequence of the measles virus, vaccine strain Schwarz, under the control of the promoter of the T7 RNA polymerase. Its size is 18967 nt.
The invention concerns also a recombinant measles virus vector comprising the above defined recombinant measles virus nucleotide sequence.
The <<rule of six>> is expressed in the fact that the total number of nucleotides present in the recombinant cDNA resulting from recombination of the cDNA sequence derived from reverse transcription of the antigenomic RNA of measles virus, and the heterologous DNA sequence finally amount to a total number of nucleotides which is a multiple of six, a rule which allows efficient replication of genome RNA of the measles virus.
A preferred recombinant measles virus vector according to the above definition is such that the heterologous DNA virus vector wherein the heterologous DNA sequence is cloned within an Additional Transcription Unit (ATU) inserted in the cDNA corresponding to the antigenomic RNA of measles virus.
The additional transcription unit (ATU) is disclosed on FIG. 2A; it can be modified provided it ultimately enables the obtained replicon in the vector to comply with the rule of six.
The location of the ATU within the cDNA derived from the antigenomic RNA of the measles virus can vary along said cDNA. It is however located in such a site that it will benefit from the expression gradient of the measles virus.
This gradient corresponds to the mRNA abundance according to the position of the gene relative to the 3′ end of the template. Accordingly, when the polymerase operates on the template (either genomic and anti-genomic RNA or corresponding cDNAs), it synthesizes more RNA made from upstream genes than from downstream genes. This gradient of mRNA abundance is however relatively smooth for measles virus. Therefore, the ATU or any insertion site suitable for cloning of the heterologous DNA sequence can be spread along the cDNA, with a preferred embodiment for an insertion site and especially in an ATU, present in the N-terminal portion of the sequence and especially within the region upstream from the L-gene of the measles virus and advantageously upstream from the M gene of said virus and more preferably upstream from the N gene of said virus.
Depending on the expression site and the expression control of the heterologous DNA, the vector of the invention allows the expression of the heterologous amino acid sequence as a fusion protein with one of the measles virus proteins.
Alternatively, the insertion site of the DNA sequence in the cDNA of the measles virus can be chosen in such a way that the heterologous DNA expresses the heterologous amino acid sequence in a form which is not a fusion protein with one of the proteins of the measles virus.
The recombinant measles virus vector according to any of the preferred definitions contains advantageously a heterologous DNA sequence which encodes a retroviral, a flaviviral amino acid sequence.
As an example, this amino acid sequence is derived from an antigen of a retrovirus selected among HIV retroviruses, or a flavivirus, especially the Yellow Fever virus or the West Nile virus.
In a particular embodiment of the invention, the heterologous amino acid sequence encoded by the recombinant measles virus vector is derived from an envelope antigen of an HIV retrovirus, especially from HIV-1.
In a preferred embodiment, this amino acid sequence encoded by the heterologous DNA sequence is selected among the gp160, the gp120 or gp41 of HIV-1, or the gp140 of HIV-1, or a mutated version of said antigens.
As one result which is expected by expressing the recombinant measles virus vector of the invention is the elicitation of an immune response, especially a humoral and/or cellular immune response, against the heterologous amino acid sequence encoded by the vector, it is preferred that the heterologous DNA sequence used is one which codes for an antigen or a mutated antigen which enables exposition of neutralizing epitopes on the produced expression product of said vector.
In a particular embodiment, the heterologous amino acid sequence expressed, can expose epitopes which are not accessible or not formed in the native antigen from which the heterologous amino acid sequence derives.
In a preferred embodiment of the invention, the heterologous DNA sequence encodes gp160ΔV3, gp160ΔV1V2, gp160ΔV1V2V3, gp140ΔV3, gp140ΔV1V2, gp140ΔV1V2V3.
Heterologous amino acid sequences are especially disclosed on FIG. 1 and can be prepared according to well-known methods starting from sequences of antigens or corresponding DNA sequences of said antigens obtained from various HIV-1 isolates.
According to a preferred embodiment of the invention, the recombinant measles virus vector is designed in such a way that the particles produced in helper cells transfected or transformed with said vector containing the DNA encoding the full length antigenomic (+)RNA of measles virus, originated from a measles virus strain adapted for vaccination, enable the production of viral particles for use in immunogenic compositions, preferably protective or even vaccine compositions.
Among measles virus strains adapted for vaccination, one can cite the Edmonston B. strain and the Schwarz strain, the latter being preferred and distributed by the company Aventis Pasteur (Lyon France) as an approved vaccination strain of measles virus.
The nucleotide sequences of the Edmonston B. strain and of the Schwarz strain, have been disclosed in WO 98/13505.
In order to prepare the recombinant measles virus vector of the invention, the inventors have designed plasmid pTM-MVSchw which contains the cDNA resulting from reverse transcription of the antigenomic RNA of measles virus and an adapted expression control sequence including a promoter and terminator for the T7 polymerase.
The recombinant measles virus vector according to the invention is preferably a plasmid.
Preferred vectors are those obtained with the nucleotide sequence of the Edmonston B. strain deposited on Jun. 12, 2002 especially:















pMV2(EdB)gp160[delta]V3HIV89.6P
CNCM I-2883



PMV2(EdB)gp160HIV89.6P
CNCM I-2884



pMV2(EdB)gp140HIV89.6P
CNCM I-2885



pMV3(EdB)gp140[delta]V3HIV89.6P
CNCM I-2886



pMV2(EdB)-NS1YFV17D
CNCM I-2887



pMV2(EdB)-EnvYFV17D
CNCM I-2888.









Other preferred vectors are those obtained with the nucleotide sequence of the Schwarz strain, deposited at the CNCM on May 26, 2003:
















pTM-MVSchw2-Es(WNV)
CNCM I-3033



pTM-MVSchw2-GFPbis-
CNCM I-3034



pTM-MVSchw2-p17p24[delta]myr(HIVB)
CNCM I-3035



pTM-MVSchw3-Tat(HIV89-6p)
CNCM I-3036



pTM-MVschw3-GFP
CNCM I-3037



pTM-MVSchw2-Es (YFV)
CNCM I-3038









and the vectors deposited at the CNCM on Jun. 19, 2003:














pTM-MVSchw2-gp140 [delta] V1 V2 V3 (HIV89-6)
CNCM I-3054


pTM-MVSchw2-gp140 [delta] V3 (HIV89-6)
CNCM I-3055


pTM-MVSchw2-gp160 [delta] V1 V2 V3 (HIV89-6)
CNCM I-3056


pTM-MVSchw2-gp160 [delta] V1 V2 (HIV89-6)
CNCM I-3057


pTM-MVSchw2-Gag SIV239 p17-p24
CNCM I-3058


[delta] myr-3-gp140 (HIV89-6)









I-2883 (pMV2(EdB)gp160[delta]V3HIV89.6P) is a plasmid derived from Bluescript containing the complete sequence of the measles virus (Edmonston strain B), under the control of the T7 RNA polymerase promoter and containing the gene of the gp160ΔV3+ELDKWAS (residues 3-9 of SEQ ID NO: 8) of the virus SVIH strain 89.6P inserted in an ATU at position 2 (between the N and P genes of measles virus). The size of the plasmid is 21264 nt.

I-2884 (pMV2(EdB)gp160HIV89.6P) is a plasmid derived from Bluescript containing the complete sequence of the measles virus (Edmonston strain B), under the control of the T7 RNA polymerase promoter and containing the gene of the gp160 of the SVIH virus strain 89.6P inserted in an ATU at position 2 (between the N and P genes of measles virus). The size of the plasmid is 21658 nt.

I-2885 (pMV2(EdB)gp140HIV89.6P) is a plasmid derived from Bluescript containing the complete sequence of the measles virus (Edmonston strain B), under the control of the T7 RNA polymerase promoter and containing the gene of the gp140 of the SVIH virus strain 89.6P inserted in an ATU at position 2 (between the N and P genes of measles virus). The size of the plasmid is 21094 nt.

I-2886 (pMV3(EdB)gp140[delta]V3HIV89.6P) is a plasmid derived from Bluescript containing the complete sequence of the measles virus (Edmonston strain B), under the control of the T7 RNA polymerase promoter and containing the gene of the gp140ΔV3(ELDKWAS; residues 3-9 of SEQ ID NO: 8) of the SVIH virus strain 89.6P inserted in an ATU at position 2 (between the N and P genes of measles virus). The size of the plasmid is 21058 nt.

I-2887 (pMV2(EdB)-NS1YFV17D) is a plasmid derived from Bluescript containing the complete sequence of the measles virus (Edmonston strain B), under the control of the T7 RNA polymerase promoter and containing the NS1 gene of the Yellow Fever virus (YFV 17D) inserted in an ATU at position 2 (between the N and P genes of measles virus). The size of the plasmid is 20163 nt.

I-2888 (pMV2(EdB)-EnvYFV17D) is a plasmid derived from Bluescript containing the complete sequence of the measles virus (Edmonston strain B), under the control of the T7 RNA polymerase promoter and containing the Env gene of the Yellow Fever virus (YFV 17D) inserted in an ATU at position 2 (between the N and P genes of measles virus). The size of the plasmid is 20505 nt.

I-3033 (pTM-MVSchw2-Es(WNV) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene of the secreted envelope, (E) of the West Nile virus (WNV), inserted in an ATU.

I-3034 (pTM-MVSchw2-GFPbis) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene of the GFP inserted in an ATU.

I-3035 (pTM-MVSchw2-p17p24[delta]myr(HIVB) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene of the gag gene encoding p17p24Δmyrproteins of the HIVB virus inserted in an ATU.

I-3036 (pTMVSchw3-Tat(HIV89-6p) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene of the Tat gene of the virus strain 89.6P inserted in an ATU.

I-3037 (pTM-MVSchw3-GFP) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain) under the control of the T7 RNA polymerase promoter and expressing the gene of the GFP gene inserted in an ATU having a deletion of one nucleotide.

1-3038 (pTM-MVSchw2-Es) (YFV) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain) under the control of the T7 RNA polymerase promoter and expressing the gene of the secreted protein of the Fever virus (YFV) inserted in an ATU.

1-3054 (pTM-MVSchw2-gp140 [delta] V1 V2 V3 (HIV89-6)) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene encoding gp140 [delta] V1 V2 (HIV 89-6) inserted in an ATU.

I-3055 (pTM-MVSchw2-gp140 [delta] V3 (HIV89-6)) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene encoding gp14 [delta] V3 (HIV 89-6) inserted in an ATU.

I-3056 (pTM-MVSchw2-gp160 [delta] V1 V2 V3 (HIV89-6)) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene encoding gp160 [delta] V1 V2 V3 (HIV 89-6) inserted in an ATU.

I-3057 (pTM-MVSchw2-gp160 [delta] V1 V2 (HIV89-6)) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene encoding gp160 [delta] V1 V2 (HIV 89-6) inserted in an ATU.

I-3058 (pTM-MVSchw2-Gag SIV239 p17-p24 [delta] myr-3-gp140 (HIV89-6)) is a plasmid derived from Bluescript containing a cDNA sequence of the complete infectious genome of the measles virus (Schwarz strain), under the control of the T7 RNA polymerase promoter and expressing the gene encoding Gag SIV239 p17-p24 [delta] myr-3-gp140 (HIV89-6) inserted in an ATU.

In a particular embodiment of the invention, the replicon contained in the recombinant measles virus vector is designed according to the map of FIG. 2 wherein <<insert>> represents the heterologous DNA sequence.
When the heterologous DNA sequence present in the recombinant measles virus vector of the invention is derived from the Yellow Fever Virus (YFV), it is advantageously selected among YFV 17D 204 commercialized by Aventis Pasteur under the trademark STAMARIL®.
When the heterologous DNA sequence present in the recombinant measles virus vector of the invention is derived from the West Nile Virus (WNV), it is advantageously selected among the neurovirulente strain IS 98-ST1.
The invention also relates to a rescue system for the assembly of recombinant measles virus expressing a heterologous amino acid sequence, which comprises a determined helper cell recombined with at least one vector suitable for expression of T7 RNA polymerase and expression of the N, P and L proteins of the measles virus transfected with a recombinant measles virus vector according to anyone of the definitions provided above.
The recombinant viruses of the invention or the VLP can also be produced in vivo by a live attenuated vaccine like MV.
The recombinant viruses of the invention or the VLP can be used in immunogenic compositions or in vaccine compositions, for the protection against RNA viruses, which antigens are expressed in the recombinant virus or in the VLP, as disclosed above and illustrated in the following examples.
The invention especially provides for immunogenic compositions or for vaccine compositions useful against HIV virus, West Nile virus or Yellow Fever virus.
The invention also concerns the use of the recombinant viruses disclosed or of the VLP, or of the recombinant vectors, for the preparation of immunogenic compositions or for the preparation of vaccine compositions.
The invention also relates to antibodies prepared against said recombinant viruses or against said VLP, especially to protective antibodies and to neutralizing antibodies. Antibodies may be polyclonal antibodies, or monoclonal antibodies.
The recombinant viruses of the invention or the VLP can be associated with any appropriate adjuvant, or vehicle which may be useful for the preparation of immunogenic compositions.
Various aspects of the invention will appear in the examples which follow and in the drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. HIV1 Env glycoprotein constructions. (A) gp160 constructions full-length and ΔV3-AAELDKWASAA (SEQ ID NO: 8), ΔV1V2 and ΔV1V2V3 (SEQ ID NO: 8) mutants (from top to bottom). The BbsI and MfeI restriction sites used to introduce the ΔV3 deletion in the other constructions are indicated. (B) gp140 constructions are the same as gp160 except that the intracytoplasmic and transmembrane regions of the gp41 have been deleted (AAELDKWASAA disclosed as SEQ ID NO: 8).
FIG. 2A. Schematic map of the pTM-MV Schw plasmid. To construct the complete sequence, the six fragments represented in the upper part were generated and recombined step by step using the unique restriction sites indicated T7=T7 promoter; hh=hammerhead ribozyme; hΔv=hepatitis delta ribozyme (=δ; T7t=T7 RNA polymerase terminator.
FIG. 2B. The pMV(+) vectors with ATU containing a green fluorescent protein (GFP) gene in position 2 and position 3. The MV genes are indicated: N (nucleoprotein), PVC (phosphoprotein and V C proteins), M (matrix), F (fusion), H (hemaglutinin), L (polymerase). T7: 17 RNA polymerase promoter; T7t: 17 RNA polymerase terminator; δ hepatitis delta virus (HDV) ribozyme; ATU: additional transcription unit.
ATU sequence: small letters represent additional sequences (copy of the N-P intergenic region of measles virus) plus cloning sites. Capital letters correspond to the inserted enhanced GFP sequence. This sequence is inserted at the SpeI site (position 3373) of the cDNA sequence of the Schwarz strain of the measles virus for ATU2 and at the SpeI site (position 9174) for the ATU3. The mutation which distinguishes normal ATU from bis(in pTM-MVSchw2-gfp and pTM-MVSchw2-GFPbis) is a substituted C (Capital letter) at the end of ATU.
FIG. 3 (A): shows that ENVHIV89.6 expression was similar for passages 2 and 5, confirming the stability of expression of transgenes in this system.
FIG. 3 (B): construction of Schwarz measles viruses (MVSchw) expressing HIV-1 antigens. (I-3054 to I-3058). Expression of HIV antigens in recombinant pTM-MVSchw.
FIG. 3(B)A: Expression of HIV-1 envelope glycoproteins in recombinant pTM-MVSchw. Vero cells were infected with the different recombinant viruses for 48H and expression of HIV Env was determined by western blot. 30 μg of each cell lysate were resolved on 4-12% SDS-PAGE, blotted onto nitrocellulose membranes and probed with a mouse monoclonal anti-HIV gp120 (Chessie, NIH) antibody. Anti-mouse IgG RPO conjugate was used as second antibody and proteins were detected using an ECL detection kit.
FIG. 3(B) B (1): Construct of double recombinant pTM-MVSchw2-Gag-3gp140, Some recombinant vectors expressing two different heterologous antigens have been constructed. They were obtained by ligation of two different recombinant pTM-MVSchw plasmids containing different inserts in position 2 and position 3. Plasmid pTM-MVSchw2-Gag-3-gp140 is shown. From this plasmid a recombinant virus was rescued that expressed both Gag and gp140 proteins (FIG. 3B(2) Western blot). Using appropriate constructions of the different inserted heterologous genes, such recombinant MV expressing two heterologous viral proteins may produce <<virus like particles>> (VLP) assembled in infected cells and secreted Gag-Env from retroviruses or prM/E from flaviviruses. Such VLP are good immunogens. Produced in vivo by a live attenuated vaccine like MV, they should be even more immunogenic.
FIG. 3BB(2): Expression of HIV-1 gp140 and SIV239 Gag in recombinant pTM-MVSchw2-GagSIV(p17-p24 [delta] myr)-3-gp140HIV. HIV gp140 and SIV Gag were detected in lysates of infected Vero cells. (A) a mouse monoclonal anti-HIV gp120 and (B) serum from macaque infected with SIVmac251.
FIG. 3C: Expression of HIV-1 Gag (p17-p24 Δmyr) in recombinant pTM-MVSchw2-GagHIV(p17-p24 [delta] myr). HIV Gag were detected in lysates of infected Vero cells with a mouse monoclonal anti-HIV Gag antibody.
FIG. 3D: Expression of HIV-1 Tat protein in recombinant pTM-MVSchw. Vero cells were infected with MVSchw-Tat HIV recombinant or control MVSchw viruses for 48H and expression of HIV Tat was determined by western blot. 30 μg of each cell lysate were resolved on 4-12% SDS-PAGE, blotted onto nitrocellulose membranes and probed with a mouse monoclonal anti-HIV Tat (BH10, NIH) antibody. Anti-mouse IgG RPO conjugate was used as second antibody and proteins were detected using an ECL detection kit.
FIG. 4. Growth kinetics of recombinant MVEdB-EnvHIV viruses on Vero cells. Cells on 35 mm dishes were infected with recombinant viruses at different MOI (as indicated). At each time point, cells were collected and cell-associated virus titers were determined using the TCID50 method on Vero cells. (A) Infections with MV EdB-tag and different MV-HIV recombinant viruses at MOI=0.0001. (B) Infections with MV2-gp160HIV at two different MOI (0.0001 and 0.01).
FIG. 5. Anti-HIV and anti-MV humoral immune responses in mice inoculated with recombinant MVEdB-EnvHIV viruses. A-B Four groups of 3 mice were immunized with 107 TCID50 of each MV-HIV recombinant virus. Antibody titers against MV (A) and HIV Env (B) were determined by ELISA in sera collected 28 days post inoculation. C-F: Anti-HIV and anti-MV antibody titers in IFNAR−/−/CD46+/− mice immunized with MV-EnvHIV viruses. (C) Anti-MV and anti-HIV titers detected 28 days after injection of increasing doses of MVEdB-gp160 (3 mice per group). (D) Anti-MV (black bars), anti-HIV (gray bars) and anti-ELDKWAS (Residues 3-9 of SEQ ID NO: 8; white bars) titers detected 28 days after injection of 5 106 TCID50 of MV-EnvHIV viruses (6 mice per group). Results are expressed as the mean values±SD.
FIG. 6. Neutralizing activities against Bx08 of sera from mice immunized with MV2-gp140HIV89.6 and MV2-gp160HIV89.6 viruses. Primary isolate Bx08 was provided by C. Moog (Strasbourg, France) and propagated once on PHA-stimulated PBMC to obtain viral stocks. 2 ng of virus was incubated for 30 min at 37° C. with 25 μl of each mouse serum (collected one month post-infection) before infection of P4R5 cells in a 96-well plate. Cells were then cultured in DMEM containing 10% of fetal calf serum until 2 days post-infection, at which time β Galactosidase activity was measured with a chemiluminescence test (Roche, Germany). Lane 1: serum of a MVEdB-Tag immunized mouse; Lane 2: serum of a MV2-gp140HIV-1 immunized mouse; Lane 3: serum of a MV2-gp160HIV-1 immunized mouse; Lane 4: non-infected cells. All assays were performed in triplicate.
FIG. 7. Edm-HIV Env vaccine candidate stimulates env-specific lymphocytes in vivo. Two groups of 3 mice were inoculated with 107 TCID50 of MV2-gp160HIV virus, and euthanized 7 day and one 1 month post inoculation. (A) ELISpot assays performed with splenocytes from immunized mice. Stimulation with HIV-gp120 purified protein (black) or irrelevant BSA (white). (B) Splenocytes collected 7 days after immunization were stimulated either with medium alone (left panel), HIV gp120 (middle panel) or EdB-tag virus (right panel). Three-color cytofluorometry detected both CD8+ (upper panel) and CD4+ (lower panel) lymphocytes producing γ-IFN after HIV gp120 and measles virus stimulations. Percentages are given according to the total CD8+ (upper panel) and CD4+ (lower panel) lymphocyte gates respectively.
FIG. 7C, D. Anti-MV and anti-HIV antibody titers in mice and macaques immunized with MV2-qp140HIV89.6 virus months after MV priming. (C) Mice (3 per group) were vaccinated with 105 TCID50 of EdB-tag MV then inoculated twice with 5 106 TCID50 of MV2-gp140HIV89.6 virus as indicated (arrows). (D) Cynomolgus macaques (#432 and 404) were vaccinated with ROUVAX® then inoculated twice with 5 106 TCID50 of MV2(gp140HIV89.6 virus as indicated (arrows).
FIG. 8. Schematic representation the additional transcription unit (ATU) (Residues 1817-1843 of SEQ ID NO: 16 and 3475-3498 of SEQ ID NO: 16, respectively, in order of appearance) and Schwarz MV vector plasmid. (A) Cis-acting elements of the ATU inserted in position 2 between phosphoprotein (P) and matrix (M) MV open reading frames. (B). Representation of the three positions of ATU insertion in the Schwarz MV vector plasmid.
FIG. 9. Expression of YFV proteins by recombinant MV. Vero cells were infected by recombinant EdB-EnvYFV and EdB-NS1YFV MV at an MOI of 0.01. Immunofluorescence was performed using a mouse polyclonal anti-YFV serum and a Cy3 secondary anti-mouse IgG antibody. All the syncytia observed in infected Vero cells were positive.
FIG. 10. YFV challenge. Six 4-weeks old mice were inoculated with a mixture of EdB-EnvYFV and EdB-NS1YFV viruses (107 TCID50) and 6 control mice were inoculated with the same dose of standard EdB-tag virus. After 1 month, anti-MV serologies were determined and a similar level of antibodies was observed in the two groups. Mice were challenged and mortality was observed.
FIG. 11. Complete nucleotide sequence of the pTM-MVSchw plasmid (CNCM I-2889; SEQ ID NO: 16). The sequence can be described as follows with reference to the position of the nucleotides:
1-8 NotI restriction site
9-28 T7 promoter
29-82 Hammer head ribozyme
83-15976 MV Schwarz antigenome
15977-16202 HDV ribozyme and 17 terminator
16203-16210 NotI restriction site
16211-16216 ApaI restriction site
16220-16226 KpnI restriction site
16226-18967 pBluescript KS(+) plasmid (Stratagene)
FIG. 12A-FIG. 12B (SEQ ID NO: 17):
The flaviral sequences which have been expressed in MV are the following:
YFV Env seq: This is the Env YFV 17D204 sequence cloned by the inventors.















pos 1 à 3
START codon



pos 4 à 51
Env signal peptide



pos 52 à 1455
Env sequence



pos 1456 à 1458
STOP codon









The stop and start codons have been added.
YFV NS1 seq: This is the NS1 YFV 17D204 sequence cloned by the inventors.















pos 1 à 3
START codon



pos 4 à 78
NS1 signal peptide



pos 79 à 1110
NS1 sequence



pos 1111 à 1113
STOP codon









The stop and start codons have been added.
FIG. 12C: WNV Env seq (SEQ ID NO: 18): this is the Env WNV sequence cloned by the inventors.















pos 1 à 3
START codon



pos 4 à 51
env signal peptide



pos 52 à 1485
Env sequence



pos 1486 à 1488
STOP codon









The stop and start codons have been added.
FIG. 12D: WNV NS1 seq (SEQ ID NO: 19): This is the NS1 WNV sequence cloned by the inventors.













pos 1 à 3
START codon


pos 4 à 78
NS1 signal peptide


pos 79 à 1104
NS1 sequence


pos 1105 à 1107
STOP codon


pos 1108 à 1110
STOP codon



(a second is added in order to respect the rule six.)









The stop and start codons have been added.
FIG. 13: Schematic representation of recombinant pTM-MVSchw-sEWNV. The MV genes are indicated: N (nucleoprotein), PVC (phosphoprotein and V, C proteins), M (matrix), F (fusion), H (hemmaglutinin), L (polymerase). T7: T7 RNA polymerase promoter; T7t: T7 RNA polymerase terminator; δ hepatitis delta virus (HDV) ribozyme; ATU: additional transcription unit.
After rescue, the recombinant virus was grown on Vero cell monolayers. The procedure used to prepare the recombinant virus was similar to the standard procedures used to prepare the live attenuated measles vaccines, except for the lyophilization that was not used.
The WNV sE expression in Vero cells infected by the MV-WN sE virus was verified by using indirect immunofluorescence assay as shown in FIG. 14.
FIG. 14: Expression of sE protein from WNV in MV induced syncytia. Immunofluorescence detection of secreted WNV Env (sE) protein in syncytia induced by recombinant MV-WN sE in Vero cells. (A, B) sE protein detected at the external surface all around recombinant MV-induced syncytia. (C, D) intracellular sE protein in recombinant MV-induced syncytia.
FIG. 15: Anti-MV serology 1 month after the first injection.
FIG. 16: HIV-1 immunogenic sequences prepared for insertion in plasmid pTM-MVSchw2 illustrated in Example II (SEQ ID NOS: 24-43, respectively, in order of appearance).



EXAMPLE I
Recombinant Measles Viruses Expressing the Native Envelope Glycoprotein of HIV1 Clade b, or Envelopes with Deleted Variable Loops, Induce Humoral and Cellular Immune Responses
Preparing a vaccine against HIV with its formidable ability at evading the host immune-responses is certainly a daunting task. However, what we have learned about the immunopathogenesis of the infection and results already obtained with animal models indicate that it may be possible (Mascola, J. R., and G. J. Nabel. 2001. Vaccines for prevention of HIV-1 disease. Immunology 13:489-495). Ideally, a preventive immunization should induce 1) antibodies that neutralize primary isolates, thereby preventing entry into host cells, and 2) CTL that eliminate the cells that were nevertheless infected. Antibodies and CTL should be directed at conserved epitopes that are critical for viral entry and replication into host cells.
Several studies, in particular with various candidate vaccines, show that a good cellular immune response might be able to control viral load, although not to eliminate the agent (Mascola, J. R., and G. J. Nabel. 2001. Vaccines for prevention of HIV-1 disease. Immunology. 13:489-495). On the other hand humoral immune responses induced so far by subunit vaccines have been disappointing, mainly because the antibodies induced did not neutralize primary isolates of HIV. For example, recombinant vaccines expressing the SIV Env were able to protect macaques against an homologous, but not an heterologous, challenge (Hu, S., et al 1996. Recombinant subunit vaccines as an approach to study correlates of protection against primate lentivirus infection. Immunology Letters. 51:115-119). DNA immunization combined with boosting with soluble recombinant gp could protect macaques against an heterologous challenge but only against a strain of SIV genetically related to the vaccine (Boyer, J. et al 1997. Protection of chimpanzees from high-dose heterologous HIV-1 challenge by DNA vaccination. Nature Medicine. 3:526-532). More recently, various <<prime-boost>> regimen, using combinations of naked DNA and viral vectors such as MVA (Amara, R. et al. 2001. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science. 292:69-74) or Adenovirus (Shiver, J. W., et al 2002. Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity. Nature. 415:331-335), gave reasonable protection against a challenge with pathogenic SHIV89.6P. <<Prime-boost>> might not be an absolute requirement since using recombinant live attenuated polio virus vaccine protected macaques against an SIV251 challenge (Crotty, S., et al 2001. Protection against simian immunodeficiency virus vaginal challenge by using Sabin poliovirus vectors. J. Virol. 75:7435-7452). It is interesting to note that in all these experiments, even when the animals were not protected against the infection, immunization caused a delay in, or even abrogated, clinical disease.
As shown by crystallography, the V1 and V2 loops of gp120 mask the CD4 binding site and the V3 loop masks the binding sites for the CXCR4 and CCR5 co-receptors (Kwong, P. D., et al 2000. Structures of HIV-1 gp120 envelope glycoproteins from laboratory-adapted and primary isolates. Structure Fold Des. 8:1329-1339; Kwong, P. D. et al 1998. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature. 393:648-659; Kwong, P. D., et al 2000. Oligomeric modeling and electrostatic analysis of the gp120 envelope glycoprotein of human immunodeficiency virus. J. Virol. 74:1961-1972). In spite of this, antibodies against the gp120 CD4 binding site are present in the sera of HIV seropositive individuals and are able to neutralize several HIV-1 isolates in in vitro tests (Burton, D. 1997. A vaccine for HIV type 1: the antibody perspective. Proceedings of the National Academy of Sciences of the United States of America. 94:10018-10023; Hoffman, T. L et al., 1999. Stable exposure of the coreceptor-binding site in a CD4-independent HIV-1 envelope protein. Proc Natl Acad Sci USA. 96:6359-6364). Also, some epitopes which are buried in the 3-D structure of the glycoprotein but become exposed after binding to the co-receptor, can induce highly neutralizing antibodies (Muster, T., et al 1993. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J. Virol. 67:6642-6647). Furthermore, neutralizing monoclonal antibodies have been obtained from patient's B cells (Parren, P. W., et ah997. Relevance of the antibody response against human immunodeficiency virus type 1 envelope to vaccine design: Immunol Lett. 57:105-112). They are directed at gp41 linear epitopes (2F5) (Muster, T., F. et al 1993. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J. Virol. 67:6642-6647), or at gp120 conformational epitopes (2G12, 17b, 48 db12) (Thali, M., et al 1993. Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding. J. Virol. 67:3978-3988; Trkola, A., et al. 1996. Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. J. Virol. 70:1100-1108). Used in synergy they can neutralize in vitro several primary isolates (Mascola, J. R. et al 1997. Potent and synergistic neutralization of human immunodeficiency virus (HIV) type 1 primary isolates by hyperimmune anti-HIV immunoglobulin combined with monoclonal antibodies 2F5 and 2G12. J. Virol. 71:7198-7206) and protect macaques against a mucosal challenge with SHIV (Baba, T. W. et al, 2000. Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection. Nat. Med. 6:200-206; Mascola, J. R., et al 1999. Protection of Macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies. J. Virol. 73:4009-4018; Mascola, J. R., et al 2000. Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat. Med. 6:207-210). However in infected people, all these antibodies are present in very low amounts, diluted in large quantities of non-neutralizing antibodies directed mainly at the antigenically variable V1, V2 and V3 gp120 loops. Therefore, there is hope that if one could induce high levels of such cross-neutralizing antibodies one may achieve at least some degree of protection. A major goal is to design a vector that will favor the production of such neutralizing antibodies.
For this reason, we engineered mutant gp160 (anchored) and gp140 (soluble) by deleting the hypervariable V1, V2 and V3 loops individually or in combination to expose conserved epitopes and induce antibodies able to neutralize primary isolates. In some of the constructions, we also replaced the V3 loop by the AAELDKWASAA (SEQ ID NO: 8) sequence, especially ELDKWAS (Residues 3-9 of SEQ ID NO: 8) sequence flanked on both sides by two Alanine to maintain the conformation of this gp41 conserved epitope normally buried in the native protein but able to induce large spectrum neutralizing antibodies (Muster, T., F. at al 1993. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J. Virol. 67:6642-6647; Binley, J. M., et al 2000. A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure. J. Virol. 74:627-643; Sanders, R. W., et al 2000. Variable-loop-deleted variants of the human immunodeficiency virus type 1 envelope glycoprotein can be stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits. J. Virol. 74:5091-5100). The normal alpha helical structure of this peptide should be conserved when exposed in our constructions at the tip of a deleted V3 loop. These constructions, in which the “immunological decoys” have been eliminated and the neutralizing epitopes have been exposed, should be good candidates for the induction of robust neutralizing antibody responses.
The HIV gp constructions were introduced into a measles vaccine vector because it induces very high titers (1/80,000) of neutralizing anti-measles antibodies. (This is probably because it replicates in a large number of cells of different types.) One may hope, therefore, that the antibody response against the engineered HIV gps will also be strong. Furthermore, measles vaccine is also a potent inducer of long lasting cellular responses. The recombinant vaccines induced cross-neutralizing antibodies as well as cellular immune responses after a single injection in CD46+/− IFN-α/β_R−/− mice. Furthermore, they induced immune responses against HIV in mice and macaques with a pre-existing anti-MV immunity.
Construction of Mutant HIV-1 Envelope Glycoproteins.
The envelope glycoproteins used in this study (FIG. 1) were derived from SHIV89.6P, a chimeric simian/human immunodeficiency virus which contains tat, rev, vpu and env genes of HIV1 in an SIVmac239 background (Reimann, K. A., et al 1996. A chimeric simian/human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate env causes an AIDS-like disease after in vivo passage in rhesus monkeys. J. Virol. 70:6922-6928). The env gene is derived from a cytopathic primary HIV1 isolate, 89.6, which is tropic for both macrophages and T cells (Collman, R., et al 1992. An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1. J. Virol. 66:7517-7521). The env sequence was amplified from the plasmid pSHIV-KB9 (NIH) that was previously cloned after in vivo passages of the original virus (Karisson, G. B., et al 1997. Characterization of molecularly cloned simian-human immunodeficiency viruses causing rapid CD4+ lymphocyte depletion in rhesus monkeys. J. Virol. 71:4218-4225). The full-length env(gp160) was amplified by PCR (Pfu polymerase) using primers that contain unique BsiWI and BssHII sites for subsequent cloning in measles vector 160E5: (5-TATCGTACGATGAGAGTGAAGGAGAAATAT-3′; SEQ ID NO: 1) and 160E3 (5′ATAGCGCGCATCACAAGAGAGTGAGCTCAA-3′; SEQ ID NO: 2). The env sequence corresponding to the secreted form (gp140) was amplified using primers 160E5 and 140E3 (5′-TATGCGCGCTTATCTTATATACCACAGCCAGT-3′; SEQ ID NO: 3). A start and a stop codon were added at both ends of the genes as well as several nucleotides after the stop codon in order to respect the “rule of six”, stipulating that the number of nucleotides of MV genome must be a multiple of 6 (Calain, P., and L. Roux. 1993. The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA. J. Virol. 67:4822-4830; Schneider, H., et at 1997. Recombinant measles viruses defective for RNA editing and V protein synthesis are viable in cultured cells. Virology. 227:314-322). Both gp160 and gp140 env fragments were cloned in PCR®2.1-TOPO® plasmid (Invitrogen) and sequenced to check that no mutations were introduced.
Mutants with loop-deletions were generated by PCR amplification of two overlapping fragments flanking the sequence to be deleted and annealing of these fragments by PCR. To replace the V3 sequence by the AAELDKWASAA (SEQ ID NO: 8) sequence containing the gp41 epitope (Muster, T., F. et al 1993. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J. Virol. 67:6642-6647), four primers were designed on both sides of Bbsl and Mfel sites encompassing the V3 sequence: ΔV3A1 (5′ATAAGACATTCAATGGATC AGGAC-3′; SEQ ID NO: 4), ΔV3A2 (5′ TGCCCATTTATCCAATTCTGCAGCATTGTTGTTGGGTCTTGTACAATT-3′; SEQ ID NO: 5), ΔV3B1 (5′-GATAAATGGGCAAGTGCTGCAAGACAAGCACATTGTAACATTGT-3′; SEQ ID NO: 6), and ΔV3B2 (5′-CTACTCCTATTGGTTCAATTCTTA-3′; SEQ ID NO: 7). The italicized sequences in ΔV3A2 and ΔV3B1 correspond to the AAELDKWASAA (SEQ ID NO: 8) epitope with a 12 nucleotides overlap. PCR amplifications with primer pairs ΔV3A1/ΔV3A2 and ΔV3B1/ΔV3B2 produced two fragments of 218 and 499 bp respectively. After gel purification, these fragments were annealed together by 15 PCR cycles without primers and amplified with ΔV3A1/ΔV3B2 primers. The resulting 705 bp fragment was cloned in PCR®2.1-TOPO® plasmid and sequenced. After digestion by Bbsl and Mfel, the fragment lacking the sequence encoding the V3 loop (ΔV3-AAELDKWASAA; SEQ ID NO: 8) was purified and introduced in place of the corresponding fragment in the gp160 and gp140 in PCR®2.1-TOPO® plasmids.
The resulting plasmids were designated pMV2-gp160ΔV3 and pMV2-gp140ΔV3.
The ΔV1V2 mutants were produced using the same procedure. Two fragments were amplified on both sides of V1V2 loop using the following primers: 160E5 (5′-TATCGTACG ATGAGAGTGAAGGAGAAATAT-3′; SEQ ID NO: 1), ΔV1V2A1 (5′-ATTTAAAGTAACACAGAGTG GGGTTAATTT-3′; SEQ ID NO: 9), ΔV1V2B1 (5′-GTTACTTTAAATTGTAACACCTCAGTCATTAC ACAGGCCTGT-3′; SEQ ID NO: 10), ΔV1V2B2 (5′-TTGCATAAAATGCTCTCCCTGGTCCTATAG-3′; SEQ ID NO: 11). The italicized sequences in ΔV1V2A1 and ΔV1V2B1 correspond to a 12 nucleotide overlap generated between the two fragments. PCR amplifications with primer pairs 160E5/ΔV1V2A1 and ΔV1V2B1/ΔV1V2B2 produced two fragments of 400 and 366 bp respectively. After gel purification, these fragments were annealed together by 15 PCR cycles without primers and amplified with 160E5/ΔV1V2B2 primers. The resulting 766 bp fragment was cloned in PCR®2.1-TOPO® plasmid and sequenced. After digestion with BsiWI (in 160E5 primer) and Bbsl, the fragment lacking the sequence encoding the V1 V2 loop was purified and introduced in place of the corresponding fragment in the gp160 and gp140 in PCR®2.1-TOPO® plasmids.
To obtain the ΔV1V2V3 mutants, the BsiWI/Bsbl fragment lacking the sequence encoding the V1 V2 loop was introduced in place of the corresponding fragment in the PCR®2.1-TOPO®-gp140ΔV3 and PCR®2.1-TOPO®-gp160ΔV3 plasmids.
After BsiWI/BssHII digestion of the different PCR®2.1-TOPO® plasmids, the native and mutant gp160 and gp140 sequences were cloned in the EdB-tag vector in ATU position 2 and ATU position 3 (FIG. 2B). The resulting plasmids were designated pMV2-gp160HIV, pMV2-gp140HIV.
Cells were maintained in Dubelbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum (FCS) for Vero cells (African green monkey kidney), or with 10% FCS, 1 mg/ml G418 for helper 293-3-46 cells (35) and for P4-CCR5 cells (Hela-CD4-CXCR4-CCR5-HIVLTR-LacZ) (12).
Recovery of Recombinant MVEdB-EnvHIV89.6 virus.
To recover the recombinant MVEdB-HIV viruses from the plasmids, the different EdB-HIV Env plasmids were used to transfect 293-3-46 helper cells.
To recover the measles virus from the EdB-HIV-Envplasmids cDNA, we used the helper-cell-based rescue system described by Radecke et al. (Radecke, F., et al 1995. Rescue of measles viruses from cloned DNA. EMBO Journal. 14:5773-5784) and modified by Parks et al. (Parks, C. L., et al 1999. Enhanced measles virus cDNA rescue and gene expression after heat shock. J. Virol. 73:3560-3566). Human helper cells stably expressing T7 RNA polymerase and measles N and P proteins (293-3-46 cells, disclosed by Radecke et al) were co-transfected using the calcium phosphate procedure with the EdB-HIV-Env plasmids (5 μg) and a plasmid expressing the MV polymerase L gene (pEMC-La, 20 ng, disclosed by Radecke et at). The virus was rescued after cocultivation of transfected 293-3-46 helper cells at 37° C. with primate Vero cells (african green monkey kidney). In this case, syncytia appeared systematically in all transfections after 2 days of coculture.
In a further experiment (FIGS. 3C-D), after overnight incubation at 37° C., the cells were heat shocked at 43° C. for 3 hours in fresh medium (40). Heat-shocked cells were incubated at 37° C. for 2 days, then transferred onto a 70% confluent Vero cells layer (10 cm Petri dishes). Syncytia appeared in Vero cells after 2-5 days of co-culture. Single syncytia were harvested and transferred to Vero cells grown in 35 mm wells. The infected cells were expanded in 75 and 150 cm3 flasks. When syncytia reached 80-90% confluence, the cells were scraped in a small volume of OptiMEM (Gibco BRL) and frozen and thawed once. After centrifugation, the supernatant, which contained virus, was stored at −80° C.
Expression of HIV1 Glycoproteins by Recombinant MV.
The rescued recombinant viruses MV2-gp140, MV2-gp160, MV3-gp140ΔV3 and MV2-gp160ΔV3 were propagated on Vero cells and the expression of HIV Env glycoproteins was analyzed by western blotting and immunofluorescence. Infection of Vera cells by recombinant MV2 viruses (with transgene insertion in position 2) showed a high expression of the HIV Env gp160 and gp140. The cleaved recombinant Env protein (gp120) was also detected. The MV3 virus (with transgene insertion in position 3) expressed lower levels of transgene, as expected due to the transcription gradient observed in MV expression. Taken together, these results indicate that HIV1 Env glycoprotein and ΔV3 mutant are efficiently expressed by the recombinant MVs.
Virus titration. The titers of recombinant MV were determined by an endpoint limit dilution assay on Vero cells. 50% tissue culture infectious dose (TCID50) were calculated using the Kärber method.
Growth Capacity of the MVEdB-EnvHIV89.6Recombinant Viruses.
To analyze the growth capacity of MVEdB-EnvHIV89.6 viruses, Vero cells were infected at different MOI (0.01 and 0.0001), incubated at 37° C., and collected at different time points. Titers of cell-associated viruses were determined for each sample using the TCID50 method on Vero cells. FIG. 4 shows that using MOI of 0.0001, the growth kinetics of MVEdB-EnvMV89.6 viruses was delayed, as compared to standard MVEdB-tag. However, using an MOI of 0.01 the production of recombinant viruses was comparable to that of standard virus, and peak titers of 107 TCID50/ml or even more were easily obtained.
In particular, monolayers of Vero cells (T-25 flasks) were infected at an MOI of 0.05 with the recombinant viruses. When syncytia reached 80-90% confluence, cells were lysed in 150 mM NaCl, 50 mM Tris pH=8, 1% NP40, 0.5 mM PMSF and 0.2 mg/ml Pefabloc (Interbiotech, France). Chromatin was removed by centrifugation and the concentration of protein in the supernatant was determined with a Bradford assay. Proteins (50 μg) were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to cellulose membranes (Amersham Pharmacia Biotech). The blots were probed with a mouse monoclonal anti-HIV gp120 antibody (Chessie 13-39.1, NIH-AIDS Research & Reference Reagent Program) or with a monoclonal anti-MV N antibody (Chemicon, Temecula, USA). A goat anti-mouse IgG antibody-horseradish peroxidase (HRP) conjugate (Amersham) was used as second antibody. Peroxidase activity was visualized with an enhanced chemiluminescence detection Kit (Pierce).
Mice Immunizations
Mice susceptible for MV infection were obtained as described previously (21). Transgenic FVB mice heterozygous for CD46 (32), the receptor for MV vaccine strains (24) were crossed with 129sv IFN-α/βR−/− mice lacking the type I interferon receptor (22). The F1 progeny was screened by PCR and the CD46+/− animals were crossed again with 129sv IFN-α/βR−/− mice. IFN-α/βR−/− CD46+/− animals were selected and used for immunization experiments. The same type of mice have already been shown to be susceptible to MV infection (20, 21).
Six-weeks-old female CD46+/− IFN-α/βR−/− mice were inoculated intraperitoneally with 107 TCID50 of MV2-gp140, MV2-gp160, MV3-gp140ΔV3 or MV2-gp160ΔV3 recombinant viruses prepared and titrated as described above. Mice were euthanized 7 days and 1 month post-infection. Spleens and whole blood were collected. Splenocytes were extracted from spleens and kept frozen in liquid nitrogen until use. Serums were decanted and serology was analyzed by ELISA for MV (Trinity Biotech, USA) and HIV (Sanofi Diagnostics, France).
Monkey Immunization
Two colony-bred rhesus macaques (Macaca mulatto) (seronegative for simian type D retrovirus, simian T-cell lymphotropic virus, simian immunodeficiency virus and MV) were vaccinated subcutaneously with 104 TCID50 of MV vaccine (ROUVAX®, Aventis Pasteur, France). They were boosted one year later by two injections of 5 106 TCID50 of MV2-gp140 recombinant virus done at 1 month interval. Blood samples were collected at different time points and anti-MV and anti-HIV antibodies were looked for.
Humoral Immune Response to Rescued Recombinant Viruses.
1stExperiment
Humoral immune responses against MV and HIV Env were analyzed by ELISA in serums collected 1 month after immunization of mice. Titers were determined by limiting dilutions. The results presented in FIG. 5 show that all the vaccinated mice responded to measles with high titers of antibodies (1/50000 to 1/80000) and to HIV Env with titers between 1/1000 and 1/5000 depending on the inserted sequence. The antibody titers between MV and HIV cannot be compared because the ELISA used have not the same sensitivity. The MV ELISA (Trinity Biotech, USA) detected the whole response against all MV proteins, while the HIV ELISA (Sanofi Diagnostics) detected only the anti-HIV Env antibodies. The capacity of these sera to neutralize a primary HIV clade B isolate was tested using indicator cells, P4R5, that express beta-galactosidase when infected with HIV (HeLa-CD4-CXCR4-CCR5-HIV LTR-LacZ cells). In preliminary experiments, we tested sera of mice immunized with recombinant MV-HIV viruses expressing native envelope glycoproteins (MV-gp160HIV-1 or MVEdB-gp140HIV89.6). The results showed that these sera had a 70-50% neutralizing activity against a primary isolate, Bx08, when used at a 1/20 dilution (FIG. 6). The neutralizing activity of sera raised against the genetically engineered Env molecules is currently under study.
2ndExperiment
In another experiment (FIG. 5C-F), sera were collected one month after immunization and heat inactivated. Anti-MV (Trinity Biotech, USA) and anti-HIV Env (Sanofi Diagnostic Pasteur, Biorad, France) antibodies were detected using commercial ELISA kits. An anti-mouse antibody-HRP conjugate (Amersham) was used as the secondary antibody. Titers were determined by limiting dilutions and calculated as the highest dilution of serum giving twice the absorbence of a 1/100 dilution of a mixture of control sera. The same ELISA kits were used for sera from macaque monkeys. An anti-monkey IgG secondary antibody was used to detect anti-HIV antibodies. Anti-MV antibodies were detected with an anti-human IgG in order to be able to calibrate the assay with standards supplied in the MV ELISA kit. They were expressed in mlU/ml. A mixture of 5 samples from negative monkeys was used as the negative control. The titer of anti-ELDKWAS (Residues 3-9 of SEQ ID NO: 8) antibodies was determined by ELISA using 96-well NeutrAvidin plates (Pierce) coated with the ELDKWAS (Residues 3-9 of SEQ ID NO: 8) biotynilated peptide (Neosystem, 5 μg/ml in NaHCO32M, Na2CO3.H2O 2M, pH 9.6). Sera from mice immunized with standard MV were used as negative controls. Peptide-bound antibodies were detected with anti-mouse antibody-HRP conjugate.
HIV-1 neutralization assays. Sera-neutralization was tested against SHIV89.6p (A. M. Aubertin, Universite Louis Pasteur, Strasbourg, H. Fleury, Bordeaux, France), 92US660, 92US714, 92HT593 (NIH-AIDS Research & Reference Reagent Program), and a clade A primary isolate: 3253 (G. Pancino, Institut Pasteur, Paris). These viruses were propagated on PHA-stimulated human PBMC as already described (42). HIV-1 neutralization assays were performed using the P4-CCR5 indicator cell line (43). P4-CCR5 cells were seeded in 96-well plates (20 000 cells per well) and incubated at 37° C. in DMEM, 10% FCS for 24 h. The medium was replaced by 100 μl DMEM, 10% FCS, DEAE dextran (100 μg/ml) and the cells were incubated at 37° C. for 30 minutes. Virus (0.5 ir 1 ng p24) was incubated with serum dilutions in 50 μl PBS at 37° C. for 20 minutes and the virus-serum mixtures were added to the cells in triplicate. After 48 hours of incubation, the β-galactosidase activity was measured using a Chemiluminescence Reporter Gene Assay (Roche, USA).
Cellular Immune Responses to Rescued Recombinant Viruses.
The capacity of splenocytes from vaccinated mice to secrete α-IFN upon in vitro stimulation was tested by flow-cytometry and ELISpot assays. Frozen cells from immunized mice were thawed 18 h before functional assays and incubated in RPMI medium supplemented with 10% 56° C.-heated FCS (Gibco) and 10 U rh-IL2 (Boehringer Mannheim). Cell viability was evaluated by trypan-blue exclusion.
To perform γ-IFN ELISpot assay, multiscreen-HA 96-wells plates were coated with capture anti-mouse γ-IFN(R4-6A2, Pharmingen) in PBS solution (6 μg/ml). After overnight incubation at 4° C., wells were washed 4 times with PBS. The remaining protein binding sites were blocked by incubating wells with 100 μl RPMI/FCS 10% for 1 h at 37° C. Medium was withdrawn just before addition of cell suspensions (100 μl) and stimulating agents (100 μl). Splenocytes from immunized mice were plated at 5.105 cell per well in duplicate in RPMI. Concanavalin A (5 μg/ml, Sigma) was used as a positive control, and RPMI/IL2 (10 U/ml) as a negative control. Cells were stimulated either with 1 μg/ml HIV1 gp120, 1 μg/ml Bovine Serum Albumin (Sigma), or Edm-Tag virus (MOI=1). After incubation for 2 h at 37° C. for viral adsorption, heated-FCS (10 μl) was added in each well (10% final concentration) and plates were incubated for 24-36 h at 37° C. To remove cells, the plates were washed twice with PBS, 4 times with PBS containing 0.05% TWEENTM 20 (Sigma), and 2 times again with PBS. For detection, a biotinylated anti-mouse γ-IFN antibody (XMG1.2, Pharmingen) was added to each well (100 μl, 4 μg/ml in PBS-0.1% FCS). After incubation for 2 h at room temperature, plates were washed 4 times with PBS-0.1% TWEENTM 20 and twice with PBS. Streptravidin-Alkaline Phosphatase (AP) conjugate (Roche) (100 μl, 1/2000 dilution in PBS) was added and incubated for 1-2 hours at room temperature. The enzyme was removed by 4 washes with PBS-0.1% TWEENTM 20 and 2 washes with PBS. Spots were then developed with BCIP/NBT color substrate (Promega) prepared in AP buffer pH 9.5 (1 M Tris, 1.5 M NaCl, 0.05 M MgCl2). Wells were monitored for spot formation by eye: after a 15-30 minutes incubation the reaction was stopped by washing under running tap water. After drying at least overnight at room temperature, colored spots were counted using an automated image analysis system ELISpot Reader (Bio-Sys).
For Flow-cytometry assays, 5 105 splenocytes (diluted in 100 μl RPMI) were stimulated in V-bottomed 96-wells plates with either 1 μg/ml HIV1 gp120 protein (AbCys) in RPMI/IL2 (10 U/ml), or EdB-tag virus (MOI=1) diluted in 100 δl RPMI/IL2. Non stimulated control cells were incubated with RPMI/IL2 (10 U/ml). After incubation for 2 h at 37° C. for viral adsorption, 10 μl FCS were added in each well (10% final concentration) and plates were incubated overnight at 37° C. The medium was then replaced by 150 μl RPMI-10% FCS containing 10 U rh-IL2 and 10 μg/ml Brefeldin A (Sigma). Cells were incubated for 4 hours at 37° C., harvested, stained with anti-mouse CD8-APC (Pharmingen) and anti-mouse CD4-CyCr (Pharmingen) for 20 minutes at room temperature, washed with PBS-BSA (0.5%), then fixed for 5 minutes at 37° C. in CytoFix (Pharmingen). After washing cells were resuspended in 100 μl PBS-BSA (0.5%) containing 0.1% Saponin (Sigma) and incubated for 30 minutes at room temperature with anti-mouse γ-IFN-PE (Pharmingen). Cells were washed again and samples were analyzed using a FACSCalibur cytometer (Becton Dickinson). The data were analyzed using Cell Quest software.
Recombinant MV Express HIV89.6 Env Glycoproteins and Replicate Efficiently.
The anchored (gp160) and soluble (gp140) forms of the HIV Env glycoprotein (strain SHIV89.6p), with or without deletion of the V3 loop and insertion of an additional ELDKWAS (Residues 3-9 of SEQ ID NO: 8) epitope, were inserted into one of the ATU of the p(+)MV vector (FIG. 2). Recombinant viruses MV2-gp140, MV2-gp160, MV3-gp140ΔV3 and MV2-gp160ΔV3 were obtained after transfection of the plasmids into the 293-3-46 helper cell line and propagation in Vero cells. MV2- and MV3- refers to the site of the insertion, position 2 or 3 respectively, of the EnvHIV89.6 construction. Expression of the EnvHIV89.6 protein was analyzed by western blotting of infected-cells lysates (FIG. 3) and immunofluorescence (not shown). The MV2-gp140 and MV2-gp160 viruses showed a high level of expression of the EnvHIV89.6 protein (FIG. 3C, lanes 1, 2, 4). As expected, the MV2-gp160Δ viruses expressed the env gp160 precursor as well as the cleaved gp120 protein (FIG. 3C, lanes 2, 4). In contrast, the MV2-gp140 and MV3-gp140ΔV3 viruses expressed only the secreted, uncleaved gp140 form. The MV3-gp140ΔV3 virus expressed slightly lower levels of transgene than viruses of the MV2-series, as expected, due to the transcription gradient observed in MV expression (FIG. 3C, lane 3). Taken together, these results indicate that EnvHIV89.6 and the ΔV3 mutants were efficiently expressed and correctly matured. The recombinant MV were passaged 5 times on Vero cells and the expression of the transgene was compared to that of the MV nucleoprotein. FIG. 3 shows that EnvHIV89.6 expression was similar for passages 2 and 5, confirming the stability of expression of transgenes in this system.
The growth of MV-EnvHIV89.6 recombinant viruses was analyzed on Vero cells using an MOI of 0.0001 or 0.01. The growth of recombinant viruses was only slightly delayed compared to that of standard EdB-tag MV rescued from p+(MV). Viruses expressing the secreted gp140 were less affected than viruses expressing the anchored gp160. The gp140ΔV3 recombinant grew at the same rate as control MV. The delay observed with viruses expressing the anchored gp160 may be due either to lower replication rate, because of the larger size of the transgene, or to reduced MV budding because of the insertion of gp160 at the surface of the infected cells. Nevertheless, the final yield of recombinant viruses was comparable to that of control MV and peak titers of about 106 to 107 TCID50/mL were obtained routinely.
Induction of Humoral Immune Response to Recombinant MV in Susceptible Mice.
The immunogenicity of MV-EnvHIV89.6 viruses was tested in genetically modified mice expressing the human CD46 MV receptor and lacking the Type I IFN receptor, increasing doses of MV2-gp160 virus (103-107 TCID50) were tested in 5 groups of 3 mice. Antibodies to MV and HIV Env were looked for by ELIA in sera collected 1 month after immunization (FIG. 5C). Both anti-MV and anti-HIV antibody titers increased when the dose of recombinant MV increased. Since high anti-MV titers were obtained when animals were inoculated with 106 to 107 TCID50, mice were immunized with 5.106 TCID50 in all further experiments. At this dose, anti-MV antibody titers were six fold higher than anti-HIV titers. One should keep in mind that immunization was against HIV Env only, whereas all MV proteins were expressed during infection. To compare the immunogenicity of the different EnvHIV constructs, four groups of 6 mice were inoculated intraperitoneally with various MV-EnvHIV89.6 viruses (FIG. 5B, 5E). All mice responded to MV (mean anti-MV titer: 5 104) and to HIV Env (mean anti-HIV titer: δ 103). No difference in anti-MV or anti-HIV or antiHIV titers was observed between the four constructs tested. Interestingly, expression from the ATU 2 or the ATU 3 position of the MV vector did not affect the antibody response. Because the ΔV3 constructions expressed an additional ELDKWAS (Residues 3-9 of SEQ ID NO: 8) epitope, the antibody response against this gp41 epitope was examined separately using a specific ELISA assay (FIG. 5F). The results showed that the ΔV3-ELDKWAS (Residues 3-9 of SEQ ID NO: 8) constructions induced higher titers of anti-ELDKWAS (Residues 3-9 of SEQ ID NO: 8) antibodies. The titer of 1/50 000 corresponds to the dilution of an immune serum capable of recognizing the antigen administered for the immunization, in ELISA assay.
MV-EnvHIV89.6 Viruses Induce Neutralizing Anti-HIV Antibodies.
The capacity of these sera to neutralize either homologous SHIV89.6p virus or various heterologous primary HIV-1 isolates was tested using a single cycle virus infectivity assay on P4-CCR5 indicator cells (43). P4-CCR5 cells express the CD4, CXCR4 and CCR5 HIV-1 receptors and have been stably transfected with an HIV LTR LacZ. Therefore, they are susceptible to HIV-1 isolates and express β-galactosidase upon infection. The sero-neutralization assay was validated using a combination of anti-HIV immunoglobulin (HIVIG) and monoclonal antibodies (2F5 and 2G12) previously shown to synergistically neutralize primary HIV isolates (17). We also used sera from infected patients that neutralize primary HIV isolates (17). We also used sera from infected patients that neutralize primary HIV primary isolates using a standard neutralization assay on human PBMCs (42). The neutralizing activity of a serum (Table 1) is expressed as the ratio of the reduction of infection obtained with this serum over the reduction obtained with negative control sera used at the same dilution (sera from HIV negative individuals and from infected patients neutralized clade B and A viruses equally well in this assay.
As shown in Table 1, antibodies induced in mice by the four MV-EnvHIV89.6 viruses neutralized the homologous SHIV89.6p at both dilutions tested (1/30 and 1/60). No significant difference was observed between the sera obtained with the different Env constructs, indicating that the secreted and anchored from of HIV glycoprotein induced neutralizing antibodies against homologous virus equally well when expressed by MV. Deleting the V3 loop, known to contain type-specific neutralizing epitopes, had no significant effect on the induction of antibodies that neutralized the homologous virus. This suggests that the deletion might have been compensated either by the addition of a second ELDKWAS (Residues 3-9 of SEQ ID NO: 8) gp41 neutralizing epitope, or by the uncovering of other neutralizing epitopes.
The antibodies induced by the recombinant viruses neutralized heterologous primary clade B isolates, except the 92HT593 isolate, as well as a clade A virus. In each case, antibodies induced by the anchored gp160 were slightly more neutralizing than antibodies induced by the secreted gp140, especially against the clade A 3253 virus. The antibodies induced by the ΔV3-ELDKWAS (Residues 3-9 of SEQ ID NO: 8) EnvHIV89.6 neutralized heterologous viruses more efficiently than those induced by the native envelope. This was particularly striking for the Bx08 virus which could be neutralized up to 90% by sera from mice immunized with MV2-gp160ΔV3 (1/30 dilution) but not by sera from mice immunized with MV expressing the native EnvHIV89.6. This neutralization was just as efficient as neutralization by positive control sera. These results show that replacing the V3 loop of EnvHIV89.6 by an additional ELDKWAS (Residues 3-9 of SEQ ID NO: 8) gp41 epitope and expressing the construct with a MV vector allowed the induction of antibodies with cross-neutralizing activity against clade A and B HIV-1 primary isolates, at least in the context of recombinant MV infection of mice.







TABLE 1







Neutralization of HIV-1 primary heterologous isolates by sera from MV-EnvHIV89.6 immunized micea.









Positive controls












Mice Sera (1/60)
Mice Sera (1/30)

Human HIV

















MW

MV2

MV2

MV2
Mab
serac


















Virus isolate
MV2
Gp140
MW
Gp160
MW
Gp140
MV2
Gp160
(2F5/2G12/
4
33-


(subtype)
Gp140
ΔV3
Gp160
ΔV3
Gp140
ΔV3
Gp160
ΔV3
HIV-IG
61/40)
1/30)





















SHIV 89.6
40
50
52
45
76 
57
72
68
ND
ND
ND


Bx08 (B)
0
31
0
40
0
76
18
90
94
94
90


92 US 660 (B)
2.5
15
13
17
ND
ND
ND
ND
ND
ND
ND


92 US 714 (B)
45
49
45
68
ND
ND
ND
ND
ND
ND
ND


92 HT 593 (B)
0
0
0
0
0
 0
 0
 0
ND
ND
ND


3253 (A)
0
0
18
30
0
10
43
49
73
54
45





aSerum was evaluated for neutralizing antibodies at two dilutions. Values are % reduction in infection of primary HIV isolates on P4-CCR5 cells in presence of mice sera (three mice per point). Determinations were made in triplicate and the standard deviations were <10%.


bMix of HIVIG (2.5 mg/ml) and Mabs 2F5 and 2G12 (25 μg/ml).


cNumbers correspond to the nomemclature used in Burrer et al.






Induction of Cellular Immune Response Against Recombinant MV
The results of these experiments performed with splenocytes from mice immunized with MV2-gp160HIV virus (FIG. 7) demonstrated that a single immunization with MV2-gp160HIV virus was able to prime HIV Env-specific lymphocytes in vivo. The γ-IFN-ELISpot assay is a sensitive method for antigen-specific cell numeration in fresh cells after in vivo immunization. This assay was used to determine whether HIV-Env-specific γ-IFN-secreting cells could be detected after a single immunization with the MV2-gp160HIV virus. FIG. 7A shows that a significant number of Env-specific cells were present in 2/3 mice tested, 7 days as well as 1 month after immunization. (For one mouse in each group the number of spots was the same after BSA or gp120 stimulation). The number of HIV-specific spots detected (up to 600/106 cells) represents 15-20% of MV-specific spots detected in the same mice (not shown), indicating that recombinant MV is able to efficiently immunize against the foreign gene expressed.
To assess the phenotype of these Env-specific cells, 3-color cytofluorometry experiments were performed on mice euthanized 7 days after immunization, at the theoretical peak of effector cells proliferation. A representative result is shown on FIG. 7B. The background γ-IFN production level for both CD4+ and CD8+ lymphocytes is shown on the left panel. For this animal, 0.09% of CD8+ lymphocytes (mean calculated for 3 mice: 0.31%) and 0.25% of CD4+ lymphocytes (mean: 0.41%) were spontaneously producing γ-IFN. The frequencies of HIV-gp120 T-cells (middle panel) in the CD8+ and CD4+ subsets were 1.76% (mean: 1.69%) and 0.92% (mean: 0.76%) respectively. It's interesting to take into account that in the same immunized mouse the frequencies of Measles specific cells in CD8+ and CD4+ subsets were 7.63% (mean: 7.03%) and 4.11% (mean: 3.50%) respectively. Indeed the recombinant MV2-gp160HIV virus expresses 6 measles proteins plus one gp160 foreign protein. Thus, the frequencies of antigen-specific lymphocytes followed the recombinant gene proportions. As a conclusion, 3-color cytofluorometry performed 7 days after MV2-gp160HIV virus vaccination showed that both CD8+ (FIG. 7B, upper panel) and CD4+ (FIG. 7B, lower panel) lymphocytes specific for HIV gp120 and measles virus were primed in vivo.
Inducing an Anti-HIV Response in Animals with Pre-Existing Anti-MV Immunity.
We first tested the possibility of boosting the anti-HIV response by a second injection of recombinant MV. Mice immunized with 5.106 TCID50 of MV2-gp140 recombinant virus (3 mice per group) were boosted with a second injection of the same recombinant MV one month after the first injection. The mean anti-MV and anti-HIV antibody titers at the time of boosting were 5 104 and 8 103 respectively. These titers increased to, respectively 5 105 and 5 104 one month after boosting. Thus, anti-MV and HIV responses can be boosted 10 times by injecting the same dose of recombinant MV one month after the first immunization.
We then tested the ability of recombinant MV to induce anti-HIV antibodies in mice and monkeys in the presence of pre-existing anti-MV immunity. Mice (3 mice per point) were first immunized with 105 TCID50 of EdB-tag MV (without an HIV insert). High levels of anti-MV antibodies were induced (FIG. 7C). The titer decreased slightly after 2 months and remained stable for the following 9 months. Mice were then inoculated with 5 106 TCID50 of MV2-gp140HIV89.6, and boosted with the same dose one month later. The titer of anti-MV antibodies was increased 100 times and high titers of anti-HIV antibodies (5 104) were induced. These titers were similar to those obtained after immunization of naive animals with two injections.
The same experiment was performed with rhesus macaques (FIG. 7D). Two macaques were immunized with a standard dose (104 TCID50) of MV vaccine (ROUVAX®, Aventis Pasteur). As for mice, high anti-MV antibody levels were induced and remained stable during one year. Macaques were then inoculated with 5 106 TCID50 of MV2-gp140HIV89.6 twice at one month interval. Anti-MV titers increased 150 times after the first injection of MV-HIV, while the second injection had no or little effect. Anti-HIV antibodies were induced by the first MV2-gp140HIV89.6 injection despite the presence of pre-existing anti-MV immunity. One month after the second MV2-gp140HIV89.6 injection, the anti-HIV antibody level had increased about 10 times and had reached titers similar to those obtained in mice. This level remained stable for the following 5 months.
The main goal of the present work was to test the immunogenicity of attenuated MV-EnvHIV recombinant viruses. We showed that such recombinants were genetically stable, expressed the HIV Env protein at high levels, and induced high titers of antibodies against both MV and the HIV Env constructs in transgenic mice. The anti-HIV antibodies titers were approximately 15-20% of those of the anti-MV antibodies. This corresponds roughly to the ratio of HIV/MV proteins expressed by the recombinant viruses. HIV Env constructions with a deleted V3 loop and an additional ELDKWAS(Residues 3-9 of SEQ ID NO: 8) gp41 epitope induced twice as much anti-ELDKWAS (Residues 3-9 of SEQ ID NO: 8) antibodies as native constructions, suggesting that the native conformation of the additional peptide was conserved in spite of its ectopic position. A high level of HIV-specific CD8+ and CD4+ cells was also induced. As much as 1.5-2% of the total CD8+ T-cells and 0.9% of the total CD4+ T-cells were HIV-specific.
However, the most important aspect of our results is that these anti-HIV antibodies were neutralizing for the homologous SHIV89.6p virus as well as for several heterologous clade A and clade B HIV-1 primary isolates. Interestingly, the anchored gp160 ΔV3-ELDKWAS (Residues 3-9 of SEQ ID NO: 8) construction induced antibodies that neutralized heterologous viruses more efficiently than those induced by the native envelope. Their neutralizing titers were similar to those of reference human HIV-neutralizing sera. The broader neutralizing capacity of these antibodies could be due either to the addition of a second ELDKWAS (Residues 3-9 of SEQ ID NO: 8) gp41 neutralizing epitope, or to the exposure of previously masked conserved neutralizing epitopes. Several groups have inserted the ELDKWAS (Residues 3-9 of SEQ ID NO: 8) epitope into various immunogenic molecules (44, 45, 46, 47). These studies showed that the conformational context in which the epitope is displayed is essential for the induction of neutralizing antibodies. A β-turn-like constraint was shown to be the most likely conformation structure of the ELDKWAS (Residues 3-9 of SEQ ID NO: 8) epitope recognized by the 2F5 neutralizing antibody (46). In our constructions, the insertion of the short AAELDKWASAA (SEQ ID NO: 8) epitope in place of the V3 loop, which is flanked by β-strands (28, 29), may have such a β-turn-like conformation.
It has been shown, already, that deleting the hyper-variable loops of HIV Env can enhance its immunogenicity (3, 48, 39). However, in previous studies neutralizing antibodies were obtained only after multiple injections of high amounts of soluble protein (23), or with a “prime boost” regimen using very large amounts of DNA and pure protein (3, 39). In contrast, we observed the same levels of neutralizing antibodies in mice injected with a single dose of MV-gp160ΔV3-ELDKWAS (Residues 3-9 of SEQ ID NO: 8). Good immunogenicity in our system results probably from the fact that the HIV Env is expressed and processed by the immune system n the same way as proteins from the live MV vaccine, a highly potent immunogen. One may hope that such levels of neutralizing antibodies could at least induce partial protection in vaccinated individuals. According to the data of others (3, 39), it might be possible to increase the immunogenicity of M-HIV Env recombinants even further by deleting the V1 and V2 loops of HIV gp120, notably to induce antibodies directed against the CD4-binding site. However, it has been recently reported that this receptor-binding site can escape from the immune response by conformational and entropic masking (49).
The presence of anti-MV immunity in nearly the entire adult human population would seem to restrict the use of MV recombinants to infants, an already worthy goal in any event. However, several studies showed that revaccinating already immunized individuals results in a boost of anti-MV antibodies, suggesting that the attenuated live vaccine replicated and expressed its proteins in spite of preexisting immunity (50). Under such circumstances, one might hope to be able to vaccinate adults against a foreign antigen with a MV recombinant. Indeed, our results demonstrate, both with mice and macaques, that high levels of anti-HIV neutralizing antibodies can be obtained in the presence of pre-existing anti-MV immunity.
Various “prime-boost” regimen, using combinations of naked DNA and viral vectors such a sMVA (1) or Adenovirus (29), gave reasonable protection against a challenge with pathogenic SHIV89.6p. In the present study, we show that a single injection of MV is able to combine humoral and cellular responses at levels similar to those induced by these complex combinations.
The same recombinants have been prepared using the cloned Schwarz strain as a vector. This should raise their immunogenicity even further.
EXAMPLE II
Construction of Schwarz Measles Viruses (MVSchw) Expressing HIV-1 Antigens
In order to test their capacity as vaccine candidates against HIV infection, we constructed several recombinant Schwarz measles viruses (MV) expressing HIV-1 antigens. Different HIV-1 genes from different open reading frames were constructed and introduced in additional transcription units in the Schwarz MV cDNA that we previously cloned (pTM-MVSchw). After rescue of the different recombinant Schwarz measles viruses, the expression of the different HIV-1 proteins was analyzed by western blotting of infected-cells lysates (FIGS. 3A-D).
Different immunogens were constructed from HIV-1 Env glycoprotein (hereafter 1-8), Gag protein (hereafter 9), and Tat protein (hereafter 10):


    1. Secreted glycoprotein gp140 from HIV-1 89.6p
    2. Anchored glycoprotein gp160 from HIV-1 89.6p
    3. Secreted glycoprotein gp140 from HIV-1 89.6p deleted from hypervariable region V3 and additional AAELDKWASAA (SEQ ID NO: 8) epitope (gp140HIV89.6ΔV3-ELDKWAS (Residues 3-9 of SEQ ID NO: 8))
    4. Anchored glycoprotein gp160 from HIV-1 89.6p deleted from hypervariable region V3 with an additional AAELDKWASAA (SEQ ID NO: 8) epitope (gp160HIV89.6ΔV3-ELDKWAS (Residues 3-9 of SEQ ID NO: 8))
    5. Secreted glycoprotein gp140 from HIV-1 89.6p deleted from hypervariable regions V1-V2 (gp140HIV89.6ΔV1V2)
    6. Anchored glycoprotein gp160 from HIV-1 89.6p deleted from hypervariable regions V1-V2 (gp160HIV89.6ΔV1V2)
    7. Secreted glycoprotein gp140 from HIV-1 89.6p deleted from hypervariable regions V1-V2-V3 (gp140HIV89.6ΔV1V2V3)
    8. Anchored glycoprotein gp160 from HIV-1 89.6p deleted from hypervariable regions V1-V2-V3 (gp160HIV89.6ΔV1 V2V3)
    9. Gag polyprotein (p17p24, delta myr) from HIV-1 (clade B consensus) truncated from the nucleoprotein ORF in C-terminal (p17p24δmyrHIV-1B)
    10. Tat protein from HIV-1 89.6p (TatHIV89.6)


The HIV env genes encoding the different forms of the Env protein were generated by PCR amplification from plasmid pSHIV-KB9 (NIH-AIDS Research & Reference Reagent Program). The specific sequences were amplified using PfuTurbo DNA polymerase (Stratagene) and specific primers. To generate the different deletions, overlapping fragments flanking the sequences to be deleted were generated and annealed together by PCR. They were then introduced by enzyme restriction cloning in place of the corresponding fragment in the gp160 and gp140 sequences already cloned in PCR®2.1-TOPO® plasmids (FIG. 1A). The different sequences generated include a start and a stop codon at both ends and respect the “rule of six”, stipulating that the nucleotides number of MV genome must be divisible by 6 (7, 8). After BsiWI/BssHII digestion, the different HIV sequences were introduced in the pTM-MVSchw vector in ATU position 2 or 3 (FIG. 1B). The resulting plasmids were designated:

    1. pTM-MVSchw2-gp140HIV 
    2. pTM-MVSchw2-gp160HIV 
    3. pTM-MVSchw2-gp140ΔV3HIV 
    4. pTM-MVSchw2-gp160ΔV3HIV 
    5. pTM-MVSchw2-gp140HIVΔV1V2
    6. pTM-MVSchw2-gp160HIVΔV1 V2
    7. pTM-MVSchw2-gp140HIVΔV1 V2V3
    8. pTM-MVSchw2-gp160HIVΔV1 V2V3
    9. pTM-MVSchw2-GagHIV(p17-p24 Δmyr)
    10. pTM-MVSchw3-TatHIV 

A recombinant virus expressing both Gag and gp140 in both positions 1 and 2 of the measles Schwarz vector was produced.

    11. pTM-MVSchw2-GagSIV239(p17-p24 Δmyr)-3-gp140HIV 

This virus expressed both proteins (Fig z). Such constructs allow the production of HIV, SHIV or SIV assembled Gag-Env “virus like particles” in cells infected by recombinant measles virus.



The HIV-1 immunogenic sequences represented in FIG. 16 have been generated:
EXAMPLE III
Recombinant Measles Viruses Expressing Different Viral Transgenes
In order to demonstrate the immunizing and protective capacities of MV as a pediatric vaccination vector, a series of recombinant measles viruses expressing different viral transgenes (listed below) from other viruses were constructed and studied. The results presented here were obtained with the old EdB-tag vector. However, we have shown that the EdB-tag was 100 times less immunogenic than the Schwarz vaccine. Thus MVEdB recombinant viruses were inoculated at higher doses. All the inserted sequences with good immunological records can be obviously inserted in the Schwarz vector.
Viral genes which have been already inserted in the recombinant measles viruses:
















HIV clade B 89.6P
gp160
gp140




gp160ΔV3
gp140ΔV3




gp160ΔV1V2
gp140ΔV1V2




gp160ΔV1V2V3
gp140ΔV1V2V3




tat










HIV clade B consensus codon optimized Gag (p17-p24)













SIV Mac 239
Nef




NefΔMyr




Nef29-236




Tat



HTLV-I
Env




Gag (p19-p24)




Tax









EXAMPLE IV
Recombinant Measles Viruses Expressing Env and NS1 from Yellow Fever Virus have Immune Capacity
Because a pediatric bivalent vaccine against measles and yellow fever should be useful, we constructed recombinant MV expressing the Env and NS1 proteins from Yellow Fever Virus (YFV 17D204, Pasteur vaccine strain) and tested their capacity to protect mice from a lethal YFV challenge.
Construction of MV-YFV Recombinant Plasmids.
The env gene was PCR amplified with Pfu polymerase using primers that contain unique BsiWI and BssHII sites for subsequent cloning in MV vector: MV-YFVEnv5 (5′-TATCGTACGATGCGAGTCGTGATTGCCCTACTG-3′; SEQ ID NO: 12) and MV-YFVEnv3 (5′-ATAGCGCGCTTATGTGTTGATGCCAACCCA-3′; SEQ ID NO: 13). The Env protein thus generated (amino acids 270-753 in YFV polyprotein) contained the signal peptide in N-terminal and a part of the transmembrane region in C-terminal. The NS1 sequence was PCR amplified in the same way with Pfu polymerase using primers: MVYFVNS5 (5′-TATCGTACGATGAGAAACA TGACAATGTCC-3′; SEQ ID NO: 14) and MVYFVNS3 (5′-ATAGGGCGCTTAATGGCTTTCATGCGTTT TCC-3′; SEQ ID NO: 15). The NS1 protein (amino acids 754-1122 in YFV polyprotein) contained its signal peptide sequence. A start and a stop codon were added at both ends of the genes as well as several nucleotides after the stop codon in order to respect the “rule of six”, stipulating that the nucleotides number of MV genome must be a multiple of 6 (7). Both env and NS1 fragments were cloned in PCR®2.1-TOPO® plasmid (Invitrogen) and sequenced to check that no mutations had been introduced. After BsiWI/BssHII digestion of the PCR®2.1-TOPO® plasmids, the env and NS1 sequences were cloned in the EdB-tag vector in ATU position 2 giving plasmids: EdB-EnvYFW and EdB-NS1YFV.
Recovery of Recombinant EdB-EnvHIV and EdB-NS1YFV Viruses.
EdB-EnvYFV and EdB-NS1YFV plasmids were used to transfect 293-3-46 helper cells as described above, and recombinant viruses were rescued from transfected cells cocultivated with Vero cells. Recombinant viruses were passaged two times on Vero cells and tested for transgene expression.
Expression of YFV Proteins by Recombinant MV.
The rescued recombinant viruses MV2-EnvYFV and MV2-NS1YFV were propagated on Vero cells and the expression of YFV proteins was analyzed by immunofluorescence. FIG. 9 shows that syncytia of Vero cells infected by recombinant MV2-YFV viruses showed a high expression of the YFV Env and NS1 proteins as detected with a mouse anti-YFV polyclonal serum. In order to determine whether the expression of YFV genes was stable, the rescued recombinant viruses were serially passaged on Vero cells. After 10 passages all the syncytia observed in infected cells were positive for YFV (not shown). Taken together, these results indicate that Env and NS1 proteins from YFV are efficiently and stably expressed over several passages by the recombinant MVs.
Mice Immunization with MV-YFV Recombinant Viruses.
A mixture of both MV2-EnvYFV and MV2-NS1YFV viruses (107 TCID50) was inoculated intraperitoneal to six CD46+/− IFN-α/βR−/− mice as described above (see MV-HIV gp experiments). As a control, six other mice received the same dose of standard measles vaccine. After one month, mice were intracranially challenged with YFV 17D204 (10 LD50 determined on FVB mice). FIG. 10 shows that 65% of MV-YFV immunized animals were fully protected against the challenge, while all animals vaccinated with standard MV died between 6 and 7 days post-challenge. Moreover, a 4-days delay in mortality was observed in mice immunized with MV-YFV, and these mice did not die with the same encephalitic clinical symptoms than mice vaccinated with standard MV vaccine. The disease was attenuated and consisted of limb paralysis. It has to be noticed that IFN-α/βR−/− mice are much more sensitive to viral infections than immunocompetent mice (102-104 times). For this reason, the lethal dose determined on immunocompetent mice was probably too high for IFN-α/βR−/− mice. The same experiment is underway using several decreasing doses of YFV challenge viruses.
In conclusion, this preliminary experiment shows that the immune responses induced by recombinant MV against YFV proteins are able to protect mice against a lethal challenge.
The above constructs were made by using the sequences disclosed on FIGS. 12A and 12B.
The same principles for the preparation of constructs would apply with sequences disclosed on FIGS. 12C and 12D.
EXAMPLE V
Vaccination Against WNV with a Live Attenuated Measles Virus (Schwarz Strain) Expressing the Secreted Form of the E Glycoprotein of the WNV (West Nile Virus)
We constructed a recombinant Schwarz measles attenuated virus expressing the WNV E soluble form and tested its capacity as vaccine candidate against WN encephalitis. The WN cDNA corresponding to the sE protein of IS-98-ST1 strain of WNV was introduced in an additional transcription unit in the Schwarz MV cDNA (pTM-MVSchw CNCM I-2889). After rescue of the recombinant Schwarz measles virus, its capacity to protect mice from a lethal WNV encephalitis following intraperitoneal challenge was tested.
A) Materials and Methods
A.1 Cells and WN Virus
The IS-98-ST1 strain of WN virus was produced on Aedes AP61 mosquito cells according to the protocol described in Desprès et al (51), Mashimo et al (52) and Lucas et al (53). The Vero-NK cell clone used in this study was selected for its capacity to fuse after infection with measles virus and to amplify the WN virus.
A.2 Titration of WN Virus on AP61 Mosquito Cells by Immunodetection of Focuses Viral Replication (Focus Immuno Assay, FIA).
The titration was performed according to the protocol described in Desprès et al (51), Mashimo et al (52) and Lucas et al (53).
The infectious titer of WN virus on AP61 cells was determined as focus forming units on AP61 cells (AP61 UFF/ml).
A.3 Purification of WN Virus Produced on AP 61 Cells.
The purification was carried out according to the protocol described in Desprès et al (51), Mashimo et al (52) and Lucas et al (53).
Briefly, the viral particles present in supernatants of AP61 cells infected during 3 days with WN virus strain IS-98-ST1 (MOI 0.4) were concentrated in 7% PEG 6000 and then purified in 30-60% discontinuous saccharose gradient and in 10-50% linear saccharose gradient. WN virious in 30% saccharose were stored at −80° C. The obtained infectious titers were about 1010 AP61 FFU/ml.
A.4 Anti-WN Antibody Detection in ELISA
The anti-WN antibody titers of diluted sera (1:100) were determined by ELISA on a given quantity of 106 AP61FFU of WN IS-98-ST1 virions purified in saccharose gradient. The protocol is described in Desprès et al (1993) and Mashimo et al (2002).
A.5 Anti-WN Immune Sera
Anti-WN immune sera were collected in adult mice genetically resistant to viral encephalitis (Mashimo et al—2002) which were tested during at least one month with intraperitoneal inoculation of 103 AP61FFU of WN virus strain IS-98-ST1.
The anti-WN antibody titer of 1:100 diluted immunsera were measured in ELISA and were about 1.4 DO units. The neutralizing titers (TNRF90) of anti-WN sera were about 1600.
Ascites of mice (HMAF) against WN strain IS-98-ST1 were obtained from animals which had been hyperimmunized with brain homogenates of baby mice inoculated with WN. The ELISA titers of anti-WN HMAF, diluted to 1:1000 were about 1 DO unit.
The anti-WN immune sera were used for indirect immunofluorescence and for passive seroprotection assays against the disease. Anti-WN HMAF were used for membrane immunodetection of viral proteins.
A6. Construction of Recombinant Schwarz Measles Virus Expressing WN sE
The WNV env gene encoding the secreted form of the protein was generated by RT-PCR amplification of viral RNA purified from viral particles (WNV IS-98-ST1 strain). The specific sequence was amplified using PfuTurbo DNA polymerase (Stratagene) and specific primers that contain unique sites for subsequent cloning in pTM-MVSchw vector: MV-WNEnv5 5′-TATCGTACGATGAGAGTTGTGTTTGTCGTGCTA-3′ (SEQ ID NO: 20; BsiWI site italicized) and MV-WNEnv3 5′-ATAGCGCGCTTAGACAGCCTTCCCAACTGA-3′ (SEQ ID NO: 21; BssHII site italicized). A start and a stop codon were added at both ends of the gene. The whole sequence generated is 1380 nucleotides long, including the start and the stop codons and respects the “rule of six”, stipulating that the nucleotides number of MV genome must be divisible by 6 [Calain, 1993 (7); Schneider, 1997 (28)]. The Env protein thus generated contains its signal peptide in N-term (18 aa) and no transmembrane region. Thus, It represents amino acids 275-732 in WNV polyprotein and has the following sequence:







(SEQ ID NO: 22)


atgagagttgtgtttgtcgtgctattgcttttggtggccccagcttacag


 


cttcaactgccttggaatgagcaacagagacttcttggaaggagtgtctg


 


gagcaacatgggtggatttggttctcgaaggcgacagcgcgtgactatca


 


tgtctaaggacaagcctaccatcgatgtgaagatgatgaatatggaggcg


 


gtcaacctggcagaggtccgcagttattgctatttggctaccgtcagcga


 


tctctccaccaaagctgcgtgcccgaccatgggagaagctcacaatgaca


 


aacgtgctgacccagcttttgtgtgcagacaaggagtggtggacaggggc


 


tggggcaacggctgcggattatttggcaaaggaagcattgacacatgcgc


 


caaatttgcctgctctaccaaggcaataggaagaaccatcttgaaagaga


 


atatcaagtacgaagtggccatttttgtccatggaccaactactgtggag


 


tcgcacggaaactactccacacaggttggagccactcaggcagggagatt


 


cagcatcactcctgcggcgccttcatacacactaaagcttggagaatatg


 


gagaggtgacagtggactgtgaaccacggtcagggattgacaccaatgca


 


tactacgtgatgactgttggaacaaagacgttcttggtccatcgtgagtg


 


gttcatggacctcaacctcccttggagcagtgctggaagtactgtgtgga


 


ggaacagagagacgttaatggagtttgaggaaccacacgccacgaagcag


 


tctgtgatagcattgggctcacaagagggagctctgcatcaagctttggc


 


tggagccattcctgtggaattttcaagcaacactgtcaagttgacgtcgg


 


gtcatttgaagtgtagagtgaagatggaaaaattgcagttgaagggaaca


 


acctatggcgtctgttcaaaggctttcaagtttcttgggactcccgcaga


 


cacaggtcacggcactgtggtgttggaattgcagtacactggcacggatg


 


gaccttgcaaagttcctatctcgtcagtggcttcattgaacgacctaacg


 


ccagtgggcagattggtcactgtcaacccttttgtttcagtggccacggc


 


caacgctaaggtcctgattgaattggaaccaccattggagactcatacat


 


agtggtgggcagaggagaacaacagatcaatcaccattggcacaagtctg


 


gaagcagcattggcaaagcctttacaaccaccctcaaaggagcgcagaga


 


ctagccgctctaggagacacagcttgggactttggatcagttggaggggt


 


gttcacctcagttgggaaggctgtctaa


 


(SEQ ID NO: 23)


MRWFWLLLLVAPAYSFNCLGMSNRDFLEGVSGATVWDLVLEGDSCVT


 


IMSKDKPTIDVKMMNMEAVNLAEVRSYCYLATVSDLSTKAACPTMGEAH


 


NDKRADPAFVCRWGWDRGWGNGCGLFGKGSIDTCAKFACSTKAIGRTI


 


LKENIKYEVAIFVHGPTTVESHGNYSTQVGATQAGRFSITPAAPSYTLKL


 


GEYGEVTVDCEPRSGIDTNAYYVMTVGTKTFLVHREWFMDLNLPWSSAGS


 


TVWRNRETLMEFEEPHATKQSVIALGSQEGALHQALAGAIPVEFSSNTVK


 


LTSGHLKCRVKMEKLQLKGTTYGVCSKAFKFLGTPADTGHGTVVLELQY


 


TGTDGPCKVPISSVASLNDLTPVGRLVTVNPFVSVATANAKVLIELEPPF


 


GDSYIVVGRGEQQINHHWHKSGSSIGKAFTTTLKGAQRLAALGDTAWDFG


 


SVGGVFTSVGKAV*






After agarose gel purification, the PCR fragment was cloned in PCR®2.1-TOPO® plasmid (Invitrogen) and sequenced to check that no mutations were introduced. After BsiWI/BssHII digestion of the PCR®2.1-TOPO® plasmid, the DNA fragment was cloned in the pTM-MVSchw vector in ATU position 2 giving plasmid pTM-MVSchw-sEWNV according to FIG. 13.
A7. Production of Recombinant Measles Virus Expressing WN sE
To recover recombinant MV from plasmid, we used the helper-cell-based rescue system described by Radecke et al. [Radecke, 1995 (35)] and modified by Parks et al. [Parks, 1999 (40)]. Human helper cells stably expressing T7 RNA polymerase and measles N and P proteins (293-3-46 cells, a kind gift from MA Billeter, University of Zurich) were transfected using the calcium phosphate procedure with pTM-MVSchw-sEWNV plasmid (5 μg) and a plasmid expressing the MV polymerase L gene (pEMC-La, 20 ng). After overnight incubation at 37° C., the transfection medium was replaced by fresh medium and a heat shock was applied (43° C. for two hours) [Parks, 1999 (40)]. After two days of incubation at 37° C., transfected cells were transferred on a CEF cells layer and incubated at 32° C. in order to avoid adaptation of the Schwarz vaccine that was originally selected on CEF cells and is currently grown on these cells. Infectious virus was recovered between 3 and 7 days following cocultivation. The recombinant virus was also rescued by the same technique after cocultivation of transfected 293-3-46 helper cells at 37° C. with Vero cells (african green monkey kidney, clone Vero-NK). In order to increase the yield of rescue and because these recombinant viruses were prepared to be used be used in mice experiments, we used Vero cells as producing cells in place of the usual chick embryo fibroblasts (CEF). Single syncytia were harvested and transferred to Vero cells grown in 35 mm wells in Dulbebecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum (FCS). The infected cells were expanded in 75 and 150 cm3 flasks. When syncytia reached 80-90% confluence (usually 36-48 hours post infection), the cells were scraped in a small volume of OptiMEM (Gibco BRL) and frozen and thawed once. After low-speed centrifugation to pellet cellular debris, the supernatant, which contained virus, was stored at −80° C. We have shown that two passages of the Schwarz virus on Vero cells did not change its immunogenic capacities in macaques.
A8. Titration of Recombinant MV-WN Virus
The titers of recombinant MV were determined by an endpoint limit dilution assay on Vero cells. 50% tissue culture infectious dose (TCID50) were calculated using the Kärber method [Karber, 1931 (41)].
A9. Immunofluorescence Detection of WNV sE Expressed in Vero Cells Infected by MV-WN sE Recombinant Virus.
The expression of the WN sE protein in cells infected by recombinant MV-WN sE was detected by immunofluorescence. Vero cells were grown on polyornithine-coated coverslips and infected by MV-WN sE at an MOI of 0.05. After two days of infection, coverslips were washed twice in PBS and fixed for 15 minutes in paraformaldehyde (4% in PBS). In some cases, cells were permeabilized by Triton X100 (0.1%, 5 min). After two PBS washes, coverslips were incubated for 15 minutes at room temperature in PBS with 2% goat serum, then incubated for 1 hour at room temperature with mouse anti-WNV immune sera or mouse anti-WNV HMAF (see A5) diluted in PBS with 2% goat serum. After washing in PBS, cells were incubated for 45 minutes at room temperature with R-phycoerythrin-conjugated goat anti-mouse IgG (SBA, Birmingham). Following washing in PBS, coverslips were mounted on slides with fluoromount (Southern Biotech Associates inc., Birmingham, Ala.).
A10, Anti-MV Antibody Detection by ELISA
Anti-MV antibodies were detected using a standard ELISA kit (Trinity Biotech, USA). An anti-mouse antibody-HRP conjugate (Amersham) was used as the secondary antibody. Titers were determined by limiting dilutions and calculated as the highest dilution of serum giving twice the absorbence of a 1/100 dilution of a mixture of control sera.
A.11 Neutralization Test by Reduction of Viral Replication Focuses (TNRF90) on VERO Cells.
Successive dilutions of sera were prepared for testing in DMEM Glutamax with 2% decomplemented FCS (Fetal Calf Serum) in tubes of 0.5 ml.
For 0.1 ml of diluted serum in DMEM Glutamax with 2% FCS, 0.1 ml of DMEM
Glutamax/2% FCS containing 100 AP61 UFF of WN virus strain IS-98-ST1 was added.
Control cell: 0.2 ml of DMEM 0.2% FCS
Control virus: 0.2 ml of DMEM Glutamax/2% FCS containing 100 AP61 UFF of WN virus strain IS-98-ST1.
2 hours with mild rotation at 37° C.
Plates with 12 cups with  ̃150 000 VERO HK cells per cup which are grown in monolayers for 24 hours in DMEM Glutamax 5% FCS
1 washing in DMEM of cell layers.
Add 0.2 ml of DMEM Glutamax/2% SVF
Add 0.2 ml of a mixture serum/WN virus on cell layers.
Incubate 2 hours at 37° C. in CO2.
Withdraw the serum/WN virus mixture of infected cell layers.
1 washing in DMEM of infected cell layers.
Add 1 ml of DMEM 2% SVF per cup.
Add 1 ml of CMC 1.6% diluted in DMEM Glutamax/2% SVF
Incubate 2 days at 37° C. in CO2.
The plaques were revealed through FIA technique. The last dilution of immunsera which neutralize at least 90 of 100 UFF of WN virus tested on VERO cells were determined (TNRF90: Test de Neutralisation par Réduction de Foyers de replication virale à 90%). The titer of neutralizing antibodies of the sera was determined by TNRF90.
A. 12 Production of WN Virus Pseudo-Particles by Cell Line MEF/3T3.Tet-Off/Pr ME.WN #h2.
Pseudo-particles of WN virus strain IS-98-ST1 composed of prME complexed glycoproteins were secreted by MEF/3T3.Tet-Off/pr ME. WN #h2 line induced for the expression of viral proteins (CNCM I-3018). They were purified for supernatants of 3-day cell culture according to the protocol used for WN virus purification. Passive seroprotection assay against WN virus in adult BALB/c mice. 6-week-old BALB/c mice were provided by the Janvier breeding Center. The dose for viral test is 100 ap61 UFF. i.e., 10 DL 50 (Tomoshi et al 2002) diluted in 100 μl of DPBS supplemented with 0.2% BSA (Bovine Serum Albumine) pH7.5 (Sigma) which are inoculated intraperitoneally. The average time for lethal effect was 10 days. Animals were observed for 2 to 3 weeks.
The sera to be tested for passive seroprotection in mice are diluted in 0.1% DPBS/0.2% BSA and inoculated 24 hours prior to viral test.
B) Results and Conclusions
B1. Production of Recombinant Measles Virus Expressing WN sE
cDNA encoding E protein of WNV strain IS-98-ST1 deleted for its transmembrane anchoring region was inserted in the genome of measles virus (Schwarz strain) according to FIG. 13.
B. 2. Preliminary Assays of Passive Seroprotection Against WN Virus in Mice
Anti-WN immune sera to be tested were obtained from mice genetically resistant to the disease (52). The anti-WN sera, late taken, were injected at dilutions 1:10 (16 TNRF90) et 1:40 (4 TNRF90) in a final volume of 0.1 ml DPBS/0.2% SAB intraperitoneally in adult BALB/c mice genetically sensitive. The antibodies were administered only 24 hours prior to the viral test or 24 hours before and 24 hours after the test with 10 DL50 of strain IS-98-ST1 of WN virus. The negative control was the injection of normal serum of mice at 1:10. The neurovirulence of WN virus was evaluated in mice tested with DPBS/0.2% SAB. The results of passive protection after two weeks of viral tests were as follows:







TABLE 1







Passive seroprotection against WNV encephalitis in adult BALB/c mice.











Passive transfer
Mortality
MDOD*






PBS/BSA (0.2%)
6\6
10.5 (±1.5)



normal serum (1:10)
6\6
12.5 (±1.5)



anti-WNV serum (1:10), 2 doses**
0\6
NA



anti-WNV serum (1:40), 2 doses
0\6
NA



anti-WNV serum (1:10), 1 dose***
1\6
12



anti-WNV serum (1:40), 1 dose
0\6
NA





(*Mean Day Of Death ± SD)


(**Day −1 and Day +1 of virus challenge)


(***Day −1 of virus challenge)






To conclude, a unique injection of anti-WN antibodies (2.5 à 10 μl of serum) obtained from mice genetically resistant to WN virus, said injection being carried out intraperitoneally in adult mice sensitive to viral encephalitis provides passive protection against a test dose.
It is noted that the sera of BALB/c mice having received anti-WN protective antibodies and resisting to viral infection have anti-WN antibody titers by ELISA which are of about 1 DO unit (for a dilution of serum of 1:100) after one month of test. This indicates that the WN virus inoculated for the test has achieved replication in protected mice, inducing a humoral response. If passive seroprotection protects against lethal encephalitis due to WN virus, it does not seem to be appropriate in order to prevent viral propagation in infected individual.
B.3. Vaccination of CD46+/− IFN-α/βR−/− Mice with MV/WN sE Virus
Mice susceptible for MV infection were obtained as described previously [Mrkic, 1998 (21)]. FVB mice heterozygous for the CD46 MV receptor transgene [Yannoutsos, 1996 (32)] were crossed with 129Sv IFN-α/βR−/− mice [Muller, 1994 (22)]. The F1 progeny was screened by PCR and the CD46+/− animals were crossed again with 129Sv IFN-α/βR−/− mice. IFN-α/βR−/− CD46+/− animals were selected and used for immunization experiments. Six-week-old CD46+/− IFN-α/βR−/− mice were inoculated intraperitoneally with a single dose of standard MV vaccine (106 TCID50, 3 mice) or MV-WN sE recombinant virus (104 or 106 TCID50, 6 mice per dose) in 300 μl phosphate buffer saline (PBS).
A serum has been taken from eye after one month of vaccination with a unique dose in order to determine the production of anti-MV, anti-WN E and neutralizing antibodies against the test virus.
b) Sera diluted to 1:100 and tested for antibodies by ELISA on purified NV virion, for:












DO unit







Ascite of anti-WN mice :
1 (control +)


Serum of anti-WN mice:
0.8 (control +)


Serum of MV vaccinated mice:
0.110 ± 0.005


Serum of MV/WN sE vaccinated mice, 104 DCIP50:
0.635 ± 0.040 (males)


Serum of MV/WN sE vaccinated mice, 104 DCIP50:
0.815 ± 0.005 



(females)


Serum of MV/WN sE vaccinated mice, 106 DCIP50:
0.800 ± 0.200 (males)


Serum of MV/WN sE vaccinated mice, 106 DCIP50:
0.900 ± 0.195 



(females)









c) In Vitro Seroneutralization Test for WNV on VERO Cells.
TNRF90 of pools of sera on 100 AP61 UFF of strain IS-98-ST1 of WN virus in VERO cells:















TNRF90









Serum of MV vaccinated mice:
<10



Serum of MV vaccinated mice MV-WN sE, 104 DCIP50:
400



Serum of MV vaccinated mice MV-WN sE, 106 DCIP50:
800










To conclude, antibodies directed against soluble E glycoprotein WN virus have the capacity to neutralize strain IS-98-ST1 used for the test by WN virus in mice in vitro.
A vaccine boost in immunized CD46+/−/IFN-α/βR−/− mice has been carried out 1 month after the beginning of vaccination with a unique dose, identical to the dose of the first injection.
After 2 weeks of boosting, sera were tested by ELISA and in TNRF90 as above:
a) Sera Diluted to 1:100 and Tested for Antibodies by ELISA on Purified WN Virion:












DO unit







Ascite of anti-WN mice :
1.4 (control +)


Serum of anti-WN mice:
1 (control +)


Serum of MV vaccinated mice:
0.110 ± 0.005


Serum of MV/WN sE vaccinated mice, 104 DCIP50:
0.810 ± 0.100 (males)


Serum of MV/WN sE vaccinated mice, 104 DCIP50:
1.150 ± 0.015 



(females)


Serum of MV/WN sE vaccinated mice, 106 DCIP50:
0.965 ± 0.230 (males)


Serum of MV/WN sE vaccinated mice, 106 DCIP50:
1.075 ± 0.240 



(females)









b) Seroneutralization Test In Vitro on VERO Cells
TNRF90 of pools of sera on 100 AP61 UFF of strain IS-98-ST1 of WN virus in VERO cells:















TNRF90









Serum of boosted MV mice:
 <10



Serum of boosted MV-WN sE, 104 DCIP50 mice:
>1600



Serum of boosted MV-WN sE, 106 DCIP50 mice:
>1600










After 4 weeks of boosting, the sera were tested by ELISA and in TNRF90 as above:
a) Sera Diluted at 1:100 and Tested for Antibodies by ELISA on Purified WN Virion:












DO unit







Ascite of anti-WN mice :
1.7 (control +)


Serum of anti-WN mice:
1.2 (control +)


Serum of MV vaccinated mice:
0.2


Serum of MV/WN sE vaccinated mice, 104 DCIP50:
1.52 (±0,15)


Serum of MV/WN sE vaccinated mice, 106 DCIP50:
1.76 (±0,10)









b) Seroneutralization In Vitro on VERO Cells
TNRF90 of pools of sera on 100 AP61 UFF of strain IS-98-ST1 of WN virus on VERO cells:












TNRF90







Serum of MV-WN sE vaccinated mice, 104 DCIP50:
 4000 (males)


Serum of MV-WN sE vaccinated mice, 104 DCIP50:
 8000 (females)


Serum of MV-WN sE vaccinated mice, 106 DCIP50:
10000-12000









To conclude, after a boost with a unique dose, the anti-WNV antibody titers and the anti-WNV neutralizing antibody titers were significantly increased by a 10-fold factor or more.
Splenocytes of CD46+/− IFN-α/βR+/− mice immunized with two injections separated by 4 weeks with the MV-WN sE virus with doses of 104 or 106DCIP50 are tested in ELISpot and flux/cytometry for the T CD4 and CD8 response after in vitro stimulation with purified viral pseudo-particles in saccharose gradients starting from supernatants of induced MEF/3T3.Tet-Off/prME.WN #h-2 (CNCM I-3018) cell line.
B.4. Passive Anti-WN Seroprotection Test in BALB/c with Anti-E Antibodies
Immune sera of CD46+/− IFN-α/βR−/− mice vaccinated with a unique dose of recombinant measles virus has been collected after one month. Various dilutions of these sera have been injected in a final volume of 0.1 ml in 6-week-old BALB/c mice and only 24 hours before inoculation of 100 AP61 UFF of strain IS-98-ST1 of WN virus (10 DL50) intraperitoneally (see protocol in §B2).
The results of passive protection after two weeks of viral test are as follows:







TABLE 2







Recombinant MV-WN sE induce antibodies that provide full


protection against WNV encephalitis in BALB/c mice











Passive transfer
Mortality
Day






PBS/BSA (0.2%)
6\6
10 to 11



anti-WNV serum (1:10), 1 dose*
0\6
NA



anti-WNV serum (1:40), 1 dose
1\6
20



anti-MV (1:10), 1 dose
4\6
10 to 11



anti-MV-WN sE 10e4 (1:10), 1 dose
3\6
 8 to 10



anti-MV-WN sE 10e6 (1:10), 1 dose
0\6
NA



anti-MV-WN sE 10e6 (1:40), 1 dose
0\6
NA



anti-MV-WN sE 10e6 (1:100), 1 dose
3\6
10 to 11





(*Day −1 of virus challenge)






To conclude, antibodies directed against WN-virus soluble glycoprotein E have the capacity to protect in vivo against WN-virus encephalitis. The vaccination of CD46+/− IFN-α/βR−/− mice with a dose of 106DCIP50 of MV-WN sE virus as a unique injection is required to induce an anti-WN E humoral response on a four-week period of time which is capable of protecting against the disease by passive seroprotection. A minimal volume of 2.5 μl of immune serum of mice vaccinated with MV-WN sE virus, is sufficient to provide a complete protection in adult BALB/c mice tested with a lethal dose of WN-virus (i.e., a ratio of about 0.1 ml of immune serum/kg). It is noted that anti-lethal sera diluted to 1:10 induce a partial protection (about 30%) against West Nile virus encephalitis.
Sera obtained in vaccinated CD46+/− IFN-α/βR−/− mice which have then been boosted with a weak dose (104 TCID50) will be tested for their capacity to provide passive protection in BALB/c mice.
B.5. Viral Test on CD46+/− IFN-α/βR−/− Mice Vaccinated with MV-WN sE
CD46−/− IFN-α/βR−/− mice vaccinated 2 months after the 2 injections of 106DCIP50 of MV-WN sE virus, these injections being done at 4 weeks internal have been tested with 100 AP61 UFF of strain IS-98-ST1 of WN virus administered intraperitoneally.
The 2 mice vaccinated with standard measles virus died the 3rd day of the test. No morbidity or lethality was observed for mice vaccinated with MV-WN sE on the 7th day of the test. To conclude, CD46+/− IFN-α/βR−/− mice immunized against soluble gpE of WN virus are protected against a lethal test dose of WN virus in the absence of anti-viral activity of alpha-interferon.
B6. New Test of Anti-WN Vaccination with an Antigen Boost
Adult CD46+/− IFN-α/βR−/− mice are vaccinated on a 4 week period of time with MV-WN sE virus at a dose of 104DCIP50 which is proposed for human and a boost with an antigen is carried out with purified pseudo-particles of WN-virus which are secreted by the cell line MEF/3T3.Tet-Off/WN prME #h2.
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## Claims

1. An expression vector for producing an infectious recombinant Schwarz strain of measles virus comprising:
A) the nucleotide sequence encoding the full length antigenomic (+)RNA strand of the measles virus Schwarz strain (from position 83 to position 15976 of SEQ ID NO: 16);
B) a T7 promoter sequence comprising a GGG motif at its 3′ end, operably linked to the nucleotide sequence of A;
C) a hammerhead ribozyme sequence (from position 29 to position 82 of SEQ ID NO: 16) located adjacent to the GGG motif at one end and adjacent to the first nucleotide of the nucleotide sequence encoding the full length anti-genomic (+)RNA strand of the measles virus Schwarz strain at the other end;
D) a T7 terminator sequence operably linked to the nucleotide sequence of A;
E) the sequence of a hepatitis delta virus ribozyme located adjacent to the last nucleotide of the nucleotide sequence encoding the full length anti-genomic (+)RNA strand of the measles virus Schwarz strain; and
F) a heterologous coding sequence encoding a heterologous amino acid sequence comprising an antigen of a heterologous RNA virus selected from a retrovirus and a flavivirus.

2. The expression vector of claim 1, wherein the infectious Schwarz strain of measles virus particles are capable of eliciting a humoral immune response, a cellular immune response, or a humoral and cellular immune response against measles virus or against said heterologous RNA virus or against both measles virus and against said heterologous RNA virus.

3. The expression vector of claim 1, comprising the nucleotide sequence of SEQ ID NO:16.

4. The expression vector of claim 1, comprising the nucleotide sequence extending from nucleotide 29 to nucleotide 16202 of the sequence of SEQ ID NO:16.

5. The expression vector of claim 1, comprising the nucleotide sequence extending from nucleotide 26 to nucleotide 16202 of the sequence of SEQ ID NO:16.

6. The expression vector of claim 1, comprising the nucleotide sequence extending from nucleotide 9 to nucleotide 16202 of the sequence of SEQ ID NO:16.

7. The expression vector of claim 1, wherein the heterologous coding sequence is cloned within the nucleotide sequence encoding the full length antigenomic (+)RNA strand of the measles virus Schwarz strain (from position 83 to position 15976 of SEQ ID NO: 16) at a position upstream of the N gene of the measles virus.

8. The expression vector of claim 1, wherein the heterologous coding sequence is cloned between the P and M gene of the measles virus.

9. The expression vector of claim 1, wherein the heterologous coding sequence is cloned between the H and L genes of the measles virus.

10. The expression vector of claim 1, wherein the antigen of a heterologous RNA virus is a West Nile Virus antigen.

11. The expression vector of claim 1, wherein the antigen of a heterologous RNA virus is Yellow Fever Virus antigen.

12. The expression vector of claim 11, wherein the Yellow Fever Virus antigen is selected from the Env antigen and the NS1 antigen.

13. The expression vector of claim 1, wherein the antigen of a heterologous RNA virus is an HIV antigen.

14. The expression vector of claim 13, wherein the HIV antigen is an envelope antigen of HIV-1 selected from gp120, gp140, and gp160.

15. The expression vector of claim 13, wherein the HIV antigen is an HIV gag antigen (p17p24).

16. The expression vector of claim 13, wherein the HIV antigen is HIV Tat antigen.

17. The expression vector of claim 13, wherein the HIV antigen is selected from the group consisting of:
A) gp160ΔV3 (SEQ ID NO: 31), in which the V3 loop consisting of amino acids 299 to 334 of SEQ ID NO: 27 is replaced by the sequence AAELDKWASAA (SEQ ID NO: 8);
B) gp160ΔV1V2 (SEQ ID NO: 35), in which the V1 and V2 loops consisting of amino acids 131 to 159 and 164 to 198 of SEQ ID NO: 27, respectively, are deleted;
C) gp160ΔV1V2V3 (SEQ ID NO: 39) in which the V1 and V2 loops consisting of amino acids 131 to 159 and 164 to 198 of SEQ ID NO: 27, respectively, are deleted and the V3 loop consisting of amino acids 299 to 334 (SEQ ID NO: 27) is replaced by the sequence AAELDKWASAA (SEQ ID NO: 8);
D) gp140ΔV3 (SEQ ID NO: 29) in which the V3 loop consisting of amino acids 299 to 334 of SEQ ID NO: 25 is replaced by the sequence AAELDKWASAA (SEQ ID NO: 8);
E) gp140ΔV1V2 (SEQ ID NO: 33) in which the V1 and V2 loops consisting of amino acids 131 to 159 and 164 to 198 of SEQ ID NO: 25, respectively, are deleted; and
F) gp140ΔV1V2V3 (SEQ ID NO: 37) in which the V1 and V2 loops consisting of amino acids 131 to 159 and 164 to 198 of SEQ ID NO: 25, respectively, are deleted and the V3 loop consisting of amino acids 299 to 334 of SEQ ID NO: 25 is replaced by the sequence AAELDKWASAA (SEQ ID NO: 8).

18. An infectious recombinant Schwarz strain of measles virus comprising a heterologous coding sequence encoding a heterologous amino acid sequence, wherein the heterologous amino acid sequence is selected from the group consisting of:
A) gp160ΔV3 (SEQ ID NO: 31), in which the V3 loop consisting of amino acids 299 to 334 of SEQ ID NO: 27 is replaced by the sequence AAELDKWASAA (SEQ ID NO: 8);
B) gp160ΔV1V2 (SEQ ID NO: 35), in which the V1 and V2 loops consisting of amino acids 131 to 159 and 164 to 198 of SEQ ID NO: 27, respectively, are deleted;
C) gp160ΔV1V2V3 (SEQ ID NO: 39) in which the V1 and V2 loops consisting of amino acids 131 to 159 and 164 to 198 of SEQ ID NO: 27, respectively, are deleted and the V3 loop consisting of amino acids 299 to 334 (SEQ ID NO: 27) is replaced by the sequence AAELDKWASAA (SEQ ID NO: 8);
D) gp140ΔV3 (SEQ ID NO: 29) in which the V3 loop consisting of amino acids 299 to 334 of SEQ ID NO: 25 is replaced by the sequence AAELDKWASAA (SEQ ID NO: 8);
E) gp140ΔV1V2 (SEQ ID NO: 33) in which the V1 and V2 loops consisting of amino acids 131 to 159 and 164 to 198 of SEQ ID NO: 25, respectively, are deleted; and
F) gp140ΔV1V2V3 (SEQ ID NO: 37) in which the V1 and V2 loops consisting of amino acids 131 to 159 and 164 to 198 of SEQ ID NO: 25, respectively, are deleted and the V3 loop consisting of amino acids 299 to 334 of SEQ ID NO: 25 is replaced by the sequence AAELDKWASAA (SEQ ID NO: 8); and
wherein the recombinant virus is infectious in Vero cells and CEF cells.

19. A recombinant vector selected from the following vectors deposited with the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES (CNCM):













pTM-MVSchw2-GFPbis-
CNCM I-3034,


pTM-MVSchw2-p17p24[delta]myr(HIVB)
CNCM I-3035,


pTM-MVSchw3-Tat(HIV89-6p)
CNCM I-3036,


pTM-MVschw3-GFP
CNCM I-3037,


pTM-MVSchw2-Es (YFV)
CNCM I-3038,


pTM-MVSchw2-gp140 [delta] V1 V2 V3(HIV89-6)
CNCM I-3054,


pTM-MVSchw2-gp140 [delta] V3(HIV89-6)
CNMC I-3055,


pTM-MVSchw2-gp160 [delta] V1 V2 V3(HIV89-6)
CNCM I-3056, and


pTM-MVSchw2-gpl 60 [delta] V1 V2(HIV89-6)
CNCM I-3057.