Patent Application: US-8336698-A

Abstract:
what is described is a recombinant poxvirus , such as vaccinia virus , containing foreign dna from plasmodium merozoite surface antigen 1 . what is also described is a vaccine containing the recombinant poxvirus for inducing an immunological response in a host animal inoculated with the vaccine .

Description:
the invention is directed to recombinant poxviruses containing therein a dna sequence from plasmodium in a nonessential region of the poxvirus genome . the recombinant poxviruses express gene products of the foreign plasmodium gene . for example , p . falciparum genes were expressed in live recombinant poxviruses . in particular , the complete msa1 gene or subfragments of the msa1 gene were isolated , characterized and inserted into vaccinia virus recombinants . enzymes and plasmids . restriction enzymes were obtained from gibco / brl , gaithersburg , md . ; new england biolabs , inc ., beverly , mass . ; and boehringer - mannheim , indianapolis , ind . t4 dna ligase , mung - bean nuclease , and dna polymerase i klenow fragment were obtained from new england biolabs , inc . standard recombinant dna techniques were used ( maniatis et al ., 1982 ) with minor modifications for cloning , screening and plasmid purification . nucleic acid sequences were confirmed using standard dideoxychain - termination reactions ( sanger et al ., 1977 ) on alkaline - denatured double - stranded plasmid templates . pibi24 and pibi25 plasmids were obtained from international biotechnologies , inc ., new haven , conn . cell lines and virus strains . a thymidine kinase mutant of the copenhagen vaccinia strain virus vp410 ( guo et al ., 1989 ) as well as a vaccinia host range mutant ( perkus et al ., 1991 ) were used to generate msa1 recombinants . all vaccinia virus stocks were produced in either vero ( atcc ccl81 ) or mrc5 ( atcc ccl71 ) or rk - 13 ( atcc ccl37 ) cells in eagles mem medium supplemented with 5 - 10 % newborn calf serum ( icn / flow laboratories , mclean , va .). oliaonucleotide - directed mutagenesis . the uracil - substituted single - stranded dna template used for the mutagenesis reactions was isolated from cj236 transformed cells . the mutations were achieved by using the protocol of kunkel et al . ( 1987 ). the various oligonucleotides were synthesized using standard chemistries ( biosearch 8700 , san rafael , calif . ; applied biosystems 380b , foster city , calif .). reconstruction of the complete msa1 gene and modification for vaccinia expression modifications of the 5 ′ extremity of the msa1 gene . referring now to fig1 the complete msa1 gene of the uganda palo - alto isolate was isolated in four fragments cloned into m13 and puc derived vectors ( chang et al ., 1988 ). the msa1 open reading frame is 5181 nucleotides long and codes for a 1726 amino acid protein . ( in all descriptions of manipulations of this gene , the adenine residue of the initiation codon will be used as nucleotide 1 and the first methionine residue as amino acid 1 .) the complete gene has been expressed under the control of the early / late h6 vaccinia promoter ( taylor et al ., 1988a , b ) by modifying the 5 ′ first 21 nucleotides ( position − 21 to − 1 ) to reconstitute the exact nucleotide sequence of the h6 promoter . referring now to fig2 this step was accomplished by cloning the drai / pvuii 520 bp fragment from clone 3 - 1 into smai digested pibi24 ; the resulting plasmid was called 24dra / pvuii . by in vitro mutagenesis ( kunkel et al ., 1987 ) using the oligonucleotide mal51 ( seq id no : 1 ) the 5 ′ extremity of the msa1 gene was adapted for expression under the control of the vaccinia h6 promoter . simultaneously , a second mutagenesis was conducted to remove two vaccinia early transcription termination signals ( yuen and moss , 1987 ) contained between position 16 to 40 of msa1 gene by using the oligonucleotide mal50 ( seq id no : 2 ). the 510 bp ecorv / bamhi fragment of mal50 + 51 was cloned into the ecorv / bglii digested sp131not plasmid . sp131not was derived from sp131 ( taylor et al ., 1991 ) by modifying the hindiii site to a noti site . the resulting plasmid was called sp131 . 5 ′ ( fig2 ). modification of the 3 ′ extremity of the msa1 gene . by using the oligonucleotides mal30 ( seq id no : 3 ) and mal31 ( seq id no : 4 ), a vaccinia early transcription termination signal was added just after the stop codon and followed by a unique restriction xhoi site . the mal30 ( seq id no : 3 ) and mal31 ( seq id no : 4 ) oligonucleotides are complementary as presented below : the psti site is localized at position 5113 in the msa1 gene . mal30 ( seq id no : 3 ) and mal31 ( seq id no : 4 ) were annealed and cloned into a psti / xhoi digested pibi24 plasmid . the resulting plasmid was called 24 ( 30 + 31 ). reconstruction of the complete msa1 gene . referring now to fig3 the 850 bp ecori / psti fragment from clone 18 - 1a was ligated into ecori / psti digested 24 ( 30 + 31 ) plasmid . the resulting plasmid was called 195eco / xho . the 3208 bp hindiii / ecori fragment from clone 3 - 1 was ligated into hindiii / ecori digested pibi24 plasmid . the resulting plasmid was called 24hind / eco . the 3910 bp ncii / hindiii fragment from 24hind / eco was ligated into mlui / hindiii digested pibi24 ( ecori − ) plasmid . the resulting plasmid was called 195hind / eco . the 820 bp ecori / xhoi fragment from 195eco / xho plasmid was ligated into ecori / xhoi digested 195hind / eco plasmid . the resulting plasmid was called 195hex ( fig3 ). referring now to fig4 the 4145 bp hindiii / acci fragment from 195hex was ligated into hindiii / nari digested sp131 . 5 ′ plasmid . the resulting plasmid was called sp131hex . the 4350 bp noti / xhoi fragment from sp131hex was ligated into the ecorv / xhoi digested vaccinia donor plasmid sd486 . the resulting plasmid was called 486195e . the 957 bp ecori fragment from clone 10 - 1 was ligated into the ecori digested plasmid 486195e . the resulting vaccinia donor plasmid carrying the complete msa1 ( p195 ) gene was called 486195 ( fig4 ). the 5210 bp nrui / xhoi fragment from 486195 was cloned into the nrui / xhoi digested mp598 plasmid ( mp598 was derived from sd494 by insertion of the vaccinia h6 promoter ( perkus et al ., 1990 ). the resulting plasmid was called atih6195 . finally , the 957 bp ecori fragment from clone 10 - 1 was ligated into the ecori digested sp131hex . the resulting plasmid was called sp131195 . the 5720 acci / xhoi fragment from sp131195 was cloned into acci / xhoi digested pibi24 plasmid . the resulting plasmid was called 24h6195 . expression of the complete msa1 gene in the vaccinia recombinant vp679 and immunization studies in rabbits construction of vaccinia virus recombinants . procedures for transfection of recombinant donor plasmids into tissue culture cells infected with a rescuing vaccinia virus and identification of recombinants by in situ hybridization on nitrocellulose filters were as previously described . ( panicali and paoletti , 1982 ; piccini et al ., 1987 ). expression of the complete msa1 gene in vp679 . vero cells were transfected with the plasmid atih6195 and infected with the rescuing virus vp410 ( guo et al ., 1989 ). the recombinant virus vp679 was isolated by successive rounds of purification as previously described ( piccini et al ., 1987 ). expression analysis of vaccinia - expressed msa1 proteins . msa1 immune rabbit serum and monoclonal antibodies have been described ( chang et al ., 1989 ). immunofluorescence and immunoprecipitation experiments of vaccinia expressed proteins and separation on sds - containing polyacrylamide gels were conducted as described ( dreyfuss et al ., 1984 ; guo et al ., 1989 ). ( 1 ) a pool of rabbit sera raised against purified p195 ( rabbit k41 , k42 , and k43 ) ( hereinafter “ rabbit serum ”); ( 2 ) ad9 . 1 and 5 . 2 — two monoclonal antibodies specific for the c - terminal part of p195 precursor and processed fragments ; and ( 3 ) ce2 . 1 — monoclonal antibody specific for the n - terminal part of p195 precursor and processed fragment . the expression of msa1 in vp679 infected cells was studied by immunofluorescence and immunoprecipitation . vero cells were infected at a moi of 0 . 2 pfu / cell and pulsed with 35 s - methionine . at 48 hours post - infection , cell lysates were harvested and immunoprecipitated with the rabbit serum . immunoprecipitated proteins were resolved on a 10 % dreyfuss gel and bands visualized by autoradiography . the msa1 polypeptide could be detected internally but not on the plasma membrane of vp679 - infected cells by immunofluorescence using the rabbit serum or the monoclonal antibodies ad9 . 1 and 5 . 2 . a weak plasma membrane fluorescence could be detected with monoclonal ce2 . 1 . by immunoprecipitation , a specific protein of an approximate molecular weight of 230 kd is recognized by the rabbit serum and by monoclonal antibodies ad9 . 1 , 5 . 2 , and ce2 . 1 . no consistent submolecular proteins could be detected indicating a lack of processing . immunological evaluation of rabbit sera . ifa and elisa titers were determined by using the procedures described by siddiqui et al . ( 1987 ) and chang et al . ( 1989 ). in vitro parasite growth inhibition was evaluated by using the procedure described by hui and siddiqui et al . ( 1987 ). results of rabbit immunization experiments with vp679 . four rabbits were immunized by intradermal route with 10 8 pfu of vp679 and boosted twice with the same dose . after the third immunization , one rabbit had an elisa titer of 6250 and the other three had lower titers . sera from each of the four rabbits reacted with plasmodium falciparum infected erythrocytes by immunofluorescence analysis . other routes of injection were tested with similar immunological responses . the first results obtained with rabbit immunization experiments demonstrated that even if some elisa titers could be achieved these titers were probably too low to be able to confer protection in the susceptible species . msa1 is the precursor of several processed proteins covering the surface of merozoites and so the complete msa1 may not be the more appropriate antigen . in an attempt to mimic the natural situation , fragments of the msa1 gene have been inserted into various vaccinia recombinants . the epstein - barr virus gp340 glycoprotein is a plasma membrane anchored protein . gp340 protein possesses at its amino terminus a consensus leader peptide , and at its carboxy terminus a consensus anchor membrane peptide ( whang et al ., 1987 ). these two ebv signal peptides have been used to express fragments of msa1 on the plasma membrane of recombinant vaccinia infected cells . the ebv gp340 gene under the control of the vaccinia h6 promoter was obtained : the gp340 5 ′ non - coding sequence ( nucleotide − 21 to − 1 ) was substituted with the same region of the h6 promoter ; at the 3 ′ extremity , after the stop codon , a vaccinia early transcription termination signal sequence was added followed by a sphi site . the resulting plasmid was called 24h6340 . first construction . the plasmid 24h6340 was digested with ecori ( position 93 of gp340 coding sequence ), treated with mung - bean nuclease , digested sphi , and ligated to the 4776 bp pvuii / sphi fragment from 24h6195 . the resulting plasmid was called 24 - i . the 4994 bp smai / xhoi fragment from 24 - i was ligated to a smai / xhoi copcs vaccinia donor plasmid ( perkus et al ., 1991 ). the resulting copcs - i plasmid was obtained and used to isolate the vaccinia recombinant vp718 . vp718 infected cells did not express any msa1 epitopes on the plasma membrane surface as detected by the rabbit serum . second construction . the 6095 bp bali / sphi fragment from 24 - i was ligated with the 163 bp scai / sphi fragment from 24h6340 . the resulting plasmid was called 24 - v . the 163 bp fragment codes for the gp340 anchor membrane domain followed by a stop codon and a vaccinia early transcription termination signal sequence . the 3197 bp smai / sphi fragment from 24 - v was ligated to a smai / sphi copcs vaccinia donor plasmid ( perkus et al ., 1991 ). the resulting plasmid was called copcs - v and used to isolate the vaccinia recombinant vp790 . vp790 infected cells did not express any msa1 epitopes on their plasma membrane surface as detected by the rabbit serum and monoclonal antibody ce2 . 1 . third construction . this construction was designed to substitute the gp340 amino leader peptide present in 24 - v by the msa1 leader peptide . the 4693 bp nrui / xbai fragment from 24 - v was ligated with the 1896 bp nrui / xbai fragment of 24h6195 . the resulting plasmid was called 24 - xvii . the 3728 bp smai / sphi fragment from 24 - xvii was ligated to a smai / sphi copcs vaccinia donor plasmid ( perkus et al ., 1991 ). the resulting plasmid was called copcs - xvii and used to generate the vaccinia recombinant vp843 . vp843 infected cells expressed msa1 epitopes on their plasma membrane surface as detected with monoclonal antibody ce2 . 1 . expression in vaccinia recombinants of c - terminal fragments of msa1 and immunization studies in rabbits construction of vaccinia donor plasmids and isolation of the corresponding vaccinia recombinants . five vaccinia recombinants expressing various parts the c - terminus of msa1 were constructed as described below . first construction . 24h6195 was digested with hindiii ( site at position 99 ) and bglii ( site at position 4676 ), the extremities were filled in with dna polymerase i klenow fragment in presence of dntps , and after gel purification , the 3850 bp fragment was ligated intramolecularly . the nucleotidic sequence of the created junction was determined by sequencing : the resulting plasmid was called 24 - xii . the 635 bp nrui / sphi fragment from 24 - xii was cloned into a copcs derived vaccinia donor plasmid ( perkus et al ., 1991 ). the resulting plasmid was called copcs - xii . the recombinant vaccinia virus expressing this construction was called vp788 . this recombinant expresses msa1 epitopes on the plasma membrane of infected cells as detected by the rabbit serum and the monoclonal antibodies ad9 . 1 and 5 . 2 . second construction . 24h6195 was cut with hindiii ( site at position 99 ) and hpai ( site at position 3702 ), the hindiii extremity was filled in with dna polymerase i klenow fragment in presence of dntps , and after gel purification , the 4508 bp fragment was ligated intramolecularly . the nucleotidic sequence of the created junction was determined by sequencing : the resulting plasmid was called 24 - xv . the 1708 bp nruii / xhoi fragment from 24 - xv was cloned into a copcs derived vaccinia donor plasmid ( perkus et al ., 1991 ). the resulting plasmid was called copcs - xv . the recombinant vaccinia virus expressing this construction was called vp806 . vp806 infected cells express msa1 epitopes on their plasma membrane as detected by the monoclonal antibody ad9 . 1 . third construction . the expression results obtained by immunoprecipitation of vp806 infected cell lysate showed the presence of a 72 kd specific protein recognized by the rabbit serum . the theoretical molecular weight of the vp806 partial msa1 protein is 64 kd . a possible glycosylation could occur at a consensus n - glycosylation site ( asn - ile - ser ; position 1613 to 1615 ) and be responsible for the observed increase of molecular weight . the putative role of the glycosylation on the immunogenecity was addressed by modifying the consensus glycosylation sequence . the 505 bp bglii / xhoi fragment of 24h6195 was cloned into a bamhi / xhoi pibi25 plasmid ; the resulting plasmid was called 25mut . by in vitro mutagenesis , the glycosylation consensus sequence was modified by using the oligonucleotide gly1 ( seq id no : 9 ). the modification was confirmed by sequencing and the resulting plasmid was called 25mut1 . the 480 bp bstbi / xhoi fragment from 25mut1 was cloned into the bstbi / xhoi digested plasmid 24 - xv . the resulting plasmid was called 24 - xv gly1 − . the 1800 bp smai / xhoi fragment from 24 - xv gly1 − was cloned into the smai / xhoi vaccinia donor plasmid copak . copak plasmid was obtained by substituting the c7l open reading by the k1l open reading frame in the copcs plasmid ( perkus et al ., 1990 ). the resulting plasmid was called copak - xv1 − . the recombinant vaccinia virus expressing this construction was called vp901 . vp901 infected cells express msa1 epitopes on their plasma membrane as detected by the rabbit serum and the monoclonal antibody ad9 . 1 . by immunoprecipitation , the specific product of vp901 infected cells recognized by the same reagents has a molecular weight of approximately 68 kd . the molecular weight difference between vp806 and vp901 expressed msa1 protein can be attributed to the modification of the glycosylation site . fourth construction . the precise localization of the peptide cleavage site in the msa1 precursor generating the c - terminal gp42 protein is known ( icopa vii conference , paris , august 1990 , poster s1 . e . 11 ). by protein sequence homology among various plasmodium falciparum strains , this site can be mapped in the uganda palo - alto msa1 precursor between the amino acids 1332 ( glu ) and 1333 ( ala ). the dna fragment coding for the gp42 c - terminal protein was obtained by pcr using the oligonucleotides c001 ( seq id no : 11 ) and c002 ( seq id no : 13 ) and the 24h6195 plasmid as template dna . the pcred dna fragment was digested by sphi and cloned into a 24h6195 hindiii filled in with dna polymerase i klenow fragment in presence of dntps and subsequently sphi digested . the resulting plasmid was called 24 - xix . the nucleotidic sequence of the created junction was determined by sequencing : the 1200 bp nrui / xhoi fragment of 24 - xix was inserted into the nrui / xhoi vaccinia donor plasmid copak h6 - 1 . the resulting plasmid was called copak xix . copak h6 - 1 was obtained by inserting the vaccinia h6 promoter in the copak plasmid . the copak xix plasmid was used to generate the vaccinia recombinant vp946 . vp946 infected cells expressed msa1 epitopes on their plasma membrane surface as demonstrated with the monoclonal antibody ad9 . 1 . fifth construction . this construction , copak - xxi , is presented in the following example 5 . results of rabbit immunization experiments with vp788 and vp806 . two rabbits were immunized by intradermal route with 10 8 pfu of vp788 or vp806 . after three boosts with the same dose , the sera were collected and analyzed by elisa , ifa , and for the rabbits immunized with vp806 , by an in vitro inhibition assay . rabbits w127 and w235 were immunized with vp788 . rabbits w292 and w293 were immunized with vp806 . elisa titers of week 11 bleedings are shown in table i . the results of the in vitro inhibition assay with rabbit sera immunized with vp806 are shown in table ii . first construction . the msa1 processed n - terminal fragment is a 83 kd protein . its n - terminal amino acid is probably the valine residue ( position 20 ) obtained after cleavage of the leader peptide . its c - terminal amino acid has never been experimentally determined , but by computer analysis ( ibi pustell sequence analysis program ; ibi , new haven , conn .) can be mapped at the amino acid 752 ( gly ). by using pcr and specific oligonucleotides , a dna fragment coding for amino acids 1 to 752 was generated and cloned into the vaccinia donor plasmid copak h6 - 1 . oligonucleotides c008 ( seq id no : 16 ) and c009 ( seq id no : 17 ) were used to amplify by pcr a 439 bp msa1 fragment ( position 1812 to 2251 ). the pcr fragment was digested with xbai and sali and ligated at xbai / sali pibi24 derived plasmid . the resulting plasmid was called 24 - 83 . the nucleotidic sequence of the 24 - 83 inserted fragment was verified . 24 - 83 was digested with styi , filled in with dna polymerase i klenow fragment in presence of dntp , digested with xhoi and subsequently ligated with the xhoi digested pcr fragment generated with oligonucleotides c001 ( seq id no : 11 ) and c002 ( seq id no : 13 ). the resulting plasmid was called 24 -( 83 + 42 ). the nucleotidic sequence flanking the restored styi site was determined : the 1590 bp xbai / sphi fragment of 24 -( 83 + 42 ) was inserted into the 4696 bp xbai / sphi fragment of 24 - xvii plasmid . the resulting plasmid was called 24 - xxi . the 3480 bp nrui / xhoi fragment of 24 - xxi was inserted into the nrui / xhoi vaccinia donor plasmid copak h6 - 1 . the resulting plasmid was called copak - xxi . 1 . chang , s . p ., hui , g . s . n ., kato , a ., siddiqui , w . a ., proc . natl . acad . sci . usa 86 , 6343 - 6347 ( 1989 ). 2 . chang , s . p ., kramer , k . j ., yamaga , k . m ., kato , a ., case , s . e ., siddiqui , w . a ., exp . para . 67 , 1 - 11 ( 1988 ). 3 . cheung , a ., leban , j ., shaw , a . r ., merkli , b ., stocker , j ., chizzolini , c ., sander , c ., perrin , l . h ., proc . natl . acad . sci . usa . 83 , 8328 - 8332 ( 1986 ). 5 . clewell , d . b . and helinski , d . r ., proc . natl . acad . sci . usa 62 , 1159 - 1166 ( 1969 ). 6 . dreyfuss , g ., adam , s . a ., choi , y . d ., mol . cell . biol . 4 , 415 - 423 ( 1984 ). 7 . guo , p ., goebel , s ., davis , s ., perkus , m . e ., languet , b ., desmettre , p ., allen , g ., paoletti , e ., j . virol . 63 , 4189 - 4198 ( 1989 ). 8 . hall , r ., hyde , j . e ., goman , m ., simmons , d . l ., hope , i . a ., mackay , j ., richle , r ., nature 311 , 279 - 392 ( 1984 ). 9 . herrera , s ., herrera , m . a ., perlaza , b . l ., burki , y ., caspers , p ., dobeli , h ., rotmann , d ., certa , u ., proc . natl . acad . sci . usa 87 , 4017 - 4021 ( 1990 ). 11 . holder , a . a ., freeman , r . r ., nicholls , s . c ., parasit . immunol . 10 , 607 - 617 ( 1988b ). 12 . hui , g . s . n ., siddiqui , w . a ., exp . para . 64 , 519 - 522 ( 1987 ). 13 . knapp , b ., shaw , a ., hundt , e ., enders , b ., kupper , h . a ., behring inst . mitt . 82 , 349 - 359 ( 1988 ). 14 . kunkel , t . a ., roberts , j . d ., zakour , r . a ., methods enzymol . 154 , 367 - 382 ( 1987 ). 15 . lyon , j . a ., haynes , j . d ., diggs , c . l ., chulay , j . d ., haidaris , c . g ., pratt - rossiter , j ., j . immunol . 138 , 895 - 901 ( 1987 ). 16 . maniatis , t ., fritsch , e . f ., sambrock , j ., in molecular cloning : a laboratory manual ( cold spring harbor laboratory , cold spring harbor , n . y .) 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