Patent Application: US-91439206-A

Abstract:
in sub - saharan africa , the vast majority of hiv transmission occurs through heterosexual contact , therefore , the initial site of hiv infection occurs within the genital tract . in a cohort of hiv - highly exposed sex workers we have identified a select group of individuals who epidemiologically and clinically appear to be hiv - resistant . studies of these women indicate a strong correlation of hiv - specific immune responses within the genital tract to protection from infection . we hypothesized that a characteristic immune phenotype is present within the genital tract of the hiv - resistant women when compared to susceptible controls . to test this we used seldi - tof mass spectrometry to profile the proteome of genital tract secretions from the hiv - resistant women and found a number of potential biomarkers which correlated to hiv - resistance . purification and tandem mass spectrometry resulted in the identification of a particular biomarker , namely trappin - 2 . this protein was tested for hiv inhibitory activity in vitro and found to be a potent inhibitor of t tropic viral infection .

Description:
unless defined otherwise , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs . although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention , the preferred methods and materials are now described . all publications mentioned hereunder are incorporated herein by reference . as used herein , “ effective amount ” refers to the administration of an amount of a given compound that achieves the desired effect . regarding trappin - 2 , a variant or bioactive fragment thereof , “ effective amount ” refers to an amount sufficient to inhibit or reduce hiv infection . as used herein , “ purified ” does not require absolute purity but is instead intended as a relative definition . for example , purification of starting material or natural material to at least one order of magnitude , preferably two or three orders of magnitude is expressly contemplated as falling within the definition of “ purified ”. as used herein , the term “ isolated ” requires that the material be removed from its original environment . as used herein , “ conservative substitution ” refers to substitution of an amino acid with an amino acid that has similar properties such that one of skill in the art would anticipate or predict that the secondary structure and hydropathic nature of the polypeptide would be substantially unchanged . as used herein , “ variant ” refers to allotypes known in the art that comprise one or more amino acid changes within a given sequence . as used herein , “ bioactive fragment ” refers to a fragment of trappin - 2 or a variant or allotype or isoform thereof which retains hiv inhibitory activity . described herein is the use of purified , isolated or synthetic trappin - 2 , variant thereof or a bioactive fragment thereof to inhibit , prevent or reduce the frequency or efficiency of human immunodeficiency virus infection . in a preferred embodiment , the hiv inhibitory peptide comprises or consists of or consists essentially of : aqepvkgpvs tkpgscpiil ircamlnppn ircamlnppn rclkdtdcpg ikkccegscg macfvpq ( seq id no : 1 ). as discussed below , this corresponds to the 6 . 6 kda peptide . in other embodiments , the hiv inhibitory peptide comprises or consists essentially of or consists of : avtgvpvk gqdtvkgrvp fngqdpvkgq vsvkgdkvk aqepvkgpvs tkpgscpiil ircamlnppn ircamlnppn rclkdtdcpg ikkccegscg macfvpq ( seq id no : 2 ). as discussed below , this corresponds to the full - length trappin - 2 peptide having had the signal sequence cleaved off . in a yet further embodiment , the hiv inhibitory peptide comprises or consists of or consists essentially of : mrassflivv vfliagtlvl eaavtgvpvk gqdtvkgrvp fngqdpvkgq vsvkgdkvk aqepvkgpvs tkpgscpiil ircamlnppn ircamlnppn rclkdtdcpg ikkccegscg macfvpq ( seq id no : 3 ). while not wishing to be bound to a particular hypothesis , as discussed below , it is believed that the hiv inhibitory peptide blocks hiv binding to t cells , for example , by binding to t cells and occupying the hiv binding site . it is of note that it is well known in the art that some modifications and changes can be made in the structure of a polypeptide without substantially altering the biological function of that peptide , to obtain a biologically equivalent polypeptide . in one aspect of the invention , the above - described peptides may include peptides that differ by conservative amino acid substitutions . the peptides of the present invention also extend to biologically equivalent peptides that differ by conservative amino acid substitutions . as used herein , the term “ conserved amino acid substitutions ” refers to the substitution of one amino acid for another at a given location in the peptide , where the substitution can be made without substantial loss of the relevant function , in this case , the folding of the epitope . in making such changes , substitutions of like amino acid residues can be made on the basis of relative similarity of side - chain substituents , for example , their size , charge , hydrophobicity , hydrophilicity , and the like , and such substitutions may be assayed for their effect on the function of the peptide by routine testing . in some embodiments , conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value ( e . g ., within a value of plus or minus 2 . 0 ), where the following may be an amino acid having a hydropathic index of about − 1 . 6 such as tyr (− 1 . 3 ) or pro (− 1 . 6 ) s are assigned to amino acid residues ( as detailed in u . s . pat . no . 4 , 554 , 101 , incorporated herein by reference ): arg (+ 3 . 0 ); lys (+ 3 . 0 ); asp (+ 3 . 0 ); glu (+ 3 . 0 ); ser (+ 0 . 3 ); asn (+ 0 . 2 ); gln (+ 0 . 2 ); gly ( 0 ); pro (− 0 . 5 ); thr (− 0 . 4 ); ala (− 0 . 5 ); his (− 0 . 5 ); cys (− 1 . 0 ); met (− 1 . 3 ); val (− 1 . 5 ); leu (− 1 . 8 ); ile (− 1 . 8 ); tyr (− 2 . 3 ); phe (− 2 . 5 ); and trp (− 3 . 4 ). in alternative embodiments , conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index ( e . g ., within a value of plus or minus 2 . 0 ). in such embodiments , each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics , as follows : ile (+ 4 . 5 ); val (+ 4 . 2 ); len (+ 3 . 8 ); phe (+ 2 . 8 ); cys (+ 2 . 5 ); met (+ 1 . 9 ); ala (+ 1 . 8 ); gly (− 0 . 4 ); thr (− 0 . 7 ); ser (− 0 . 8 ); trp (− 0 . 9 ); tyr (− 1 . 3 ); pro (− 1 . 6 ); his (− 3 . 2 ); glu (− 3 . 5 ); gin (− 3 . 5 ); asp (− 3 . 5 ); asn (− 3 . 5 ); lys (− 3 . 9 ); and arg (− 4 . 5 ). in alternative embodiments , conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class , where the amino acids are divided into non - polar , acidic , basic and neutral classes , as follows : non - polar : ala , val , len , lie , phe , trp , pro , met ; acidic : asp , glu ; basic : lys , arg , his ; neutral : gly , ser , thr , cys , asn , gln , tyr . accordingly , in some embodiments , the hiv inhibitory peptide is derived from trappin - 2 , that is , the hiv inhibitory peptide comprises a peptide that has at least 60 % or at least 65 % or at least 70 % or at least 75 % or at least 80 % or at least 81 %, or at least 82 %, or at least 83 % or at least 84 % or at least 85 % or at least 86 % or at least 87 % or at least 88 % or at least 89 % or at least 90 % or at least 91 % or at least 92 % or at least 93 % or at least 94 % or at least 95 % identity with seq id no : 1 , for example , with amino acids 1 - 67 of seq id no . 1 and retains hiv inhibitory activity , as discussed below . as will be appreciated by one of skill in the art , in some embodiments , the hiv inhibitory peptide as described above may be genetically or chemically fused to a carrier protein . that is , the hiv inhibitory peptide may be crosslinked to a carrier protein or a nucleic acid molecule encoding the hiv inhibitory peptide may be inserted into an expression system such that the hiv inhibitory peptide is flanked at either its native n terminal or c terminal or both non - native amino acid residues . it is of note that in addition to being an hiv inhibitory peptide , the above - described peptides are also immunotherapeutic agents in that they will modify the immune response to hiv or alter targeted cells of infection ( which are immune cells ) such that they are rendered impermeable or substantially impermeable to the virus and / or are no longer able to support viral replication . in other embodiments , there is provided an expression system comprising a nucleic acid deduced from the amino acid sequence of any one of the above - described peptides , preferably seq id no : 1 or a variant or bioactive fragment thereof , operably linked to a suitable promoter . as will be appreciated by one of skill in the art , the suitable promoter may be for expression and recovery of the peptide , for example , in yeast , baculovirus or other suitable expression systems , or a strong inducible or constitutive host - specific promoter for construction of gene therapy or gene replacement vectors or suitable promoters for transient expression . in yet other embodiments , the hiv inhibitory peptide as described herein may be fused to known peptide domains , for example , for targeting ( signal domains or targeting domains ) or purification ( affinity domains ) of the peptides . such chimeric peptides are within the scope of the invention . in other embodiments , the purified , isolated or synthesized peptide as described above is administered to an individual in need of such treatment for inhibiting or reducing human immunodeficiency virus infection . in yet other embodiments , the purified , isolated or synthesized peptide as described above is used in the manufacture of a pharmaceutical composition for inhibiting or reducing human immunodeficiency virus infection . in a preferred embodiment , the peptide is the active agent in a composition arranged to be applied vaginally , as discussed herein . in yet other embodiments , the purified , isolated or synthesized peptide as described above is used in the manufacture of a microbicide , as discussed herein . as will be appreciated by one of skill in the art , in these embodiments , one or more of the hiv inhibitory peptides may be combined with known microbicides effective against common vaginal tract pathogens . as discussed below , in these embodiments , the medicament comprising one or more of the hiv inhibitory peptides may be arranged for topical administration . in some embodiments , one or more of the peptides described above may be combined with a pharmaceutically or pharmacologically acceptable carrier , excipient or diluent , either biodegradable or non - biodegradable . exemplary examples of carriers include , but are by no means limited to , for example , poly ( ethylene - vinyl acetate ), copolymers of lactic acid and glycolic acid , poly ( lactic acid ), gelatin , collagen matrices , polysaccharides , poly ( d , l lactide ), poly ( malic acid ), poly ( caprolactone ), celluloses , albumin , starch , casein , dextran , polyesters , ethanol , mathacrylate , polyurethane , polyethylene , vinyl polymers , glycols , mixtures thereof and the like . standard excipients include gelatin , casein , lecithin , gum acacia , cholesterol , tragacanth , stearic acid , benzalkonium chloride , calcium stearate , glyceryl monostearate , cetostearyl alcohol , cetomacrogol emulsifying wax , sorbitan esters , polyoxyethylene alkyl ethers , polyoxyethylene castor oil derivatives , polyoxyethylene sorbitan fatty acid esters , polyethylene glycols , polyoxyethylene stearates , colloidol silicon dioxide , phosphates , sodium dodecylsulfate , carboxymethylcellulose calcium , carboxymethylcellulose sodium , methylcellulose , hydroxyethylcellulose , hydroxypropylcellulose , hydroxypropylmethycellulose phthalate , noncrystalline cellulose , magnesium aluminum silicate , triethanolamine , polyvinyl alcohol , polyvinylpyrrolidone , sugars and starches . see , for example , remington : the science and practice of pharmacy , 2000 , gennaro , a r ed ., eaton , pa . : mack publishing co . as will be apparent to one knowledgeable in the art , specific carriers and carrier combinations known in the art may be selected based on their properties and release characteristics in view of the intended use . specifically , the carrier may be ph - sensitive , thermo - sensitive , thermo - gelling , arranged for sustained release or a quick burst . in some embodiments , carriers of different classes may be used in combination for multiple effects , for example , a quick burst followed by sustained release . in some embodiments , one or more of the hiv inhibitory peptides described above in any suitable form as described above may be combined with biological or synthetic targeting molecules , for example , site - specific binding proteins , antibodies , lectins or ligands , for targeting the hiv inhibitory peptides to a specific region or location . in alternative embodiments , the hiv inhibitory peptides may be formulated for administration to a specific region of the body , for example , vaginally . that is , in these embodiments , the hiv inhibitory peptide is combined with a suitable carrier or excipient such that the medicament is arranged for vaginal administration . it is of note that methods for preparation of medicaments for vaginal application are well known in the art , see for example u . s . pat . no . 6 , 432 , 440 which is incorporated herein by reference for this purpose . in other embodiments , the hiv inhibitory peptide may be combined with other known treatments as a form of joint therapy . for example , the hiv inhibitory peptide may be combined with other anti - hiv compounds , for example , azidothymidine ( azt ), lamivudine ( 3tc ), dideoxyinosine ( ddi ), dideoxycytidine ( ddc ) and ritonavir , as well as other reverse transcriptase and protease inhibitors . we describe here the discovery of a previously uncharacterized human protein that inhibits hiv infection in an in vitro model . importantly , this protein was discovered in hiv resistant women with its elevated expression tightly associated with hiv - resistance . hiv - resistance in humans is a very rare phenomenon with only a handful of groups claiming to have identified and studied these individuals . the cohort we have characterized among the best characterized in the world . in addition , while mechanisms of hiv - resistance have been described ( ie . the ccr5 32 deletion in caucasian individuals ) no known mechanism of resistance has been correlated to the women within this study group to date . given the high exposure rates through heterosexual contact and the fact that once infected with hiv , it is impossible to clear , we hypothesized that these hiv resistant women are protected from infection at the genital mucosal barrier , thereby preventing infection from ever occurring . the discovery of a protein that is elevated in hiv - resistant women , expressed in the genital tract mucosa , and directly inhibits hiv - infection , lends strong support to our hypothesis . this is an important finding as no other inhibitor expressed at the genital tract level ( ie . beta - chemokines or sdf - 1 , slp - 1 , lactoferrin etc . . . . ) has ever correlated with hiv - resistance in humans . interestingly , the mechanism by which trappin - 2 seems to be inhibiting hiv - infection is novel when compared to other known inhibitors , including the related protein slp - 1 , as discussed below . as discussed herein , the protein can be used as a preventative measure / therapeutic intervention for hiv infection . as discussed herein , the peptide may be used for example either as an active ingredient in a microbicide or as an adjuvant or component of a therapeutic or preventative mucosal based vaccine . the united nations has modeled the effective distribution and use of a hypothetical microbicide and shown that this preventative measure would save millions of lives every year . as the initial site of infection with hiv in this cohort is the genital tract , it seems logical that mechanisms involved in mediating protection would be evident at the genital mucosa . to investigate potential mechanisms of protection against hiv we employed a novel approach to screen the proteome of genital tract secretions from hiv - resistant sex - workers and comparison groups . the protein content of cervical lavage ( cvl ) samples was analyzed using surface - enhanced laser desorption / ionization time - of - flight mass spectrometry ( seldi - tof ms ). this approach uses proteinchip arrays capable of specifically capturing proteins based on their chemical properties onto chromatographic surfaces . bound proteins are then detected and characterized by linear time - of - flight mass spectrometry . protein ( s ) of interest are then purified , sequenced and identified by tandem mass spectrometry . this strategy has advantages for study of mucosal material with limited sample volume in that it can quantitatively detect multiple proteins in a complex sample , identify unknown or unsuspected proteins , has high sample through - put and is highly sensitive ( 8 , 9 ). the genital tract proteome of hiv - resistant sex workers was compared to hiv uninfected women from the same sex worker cohort , with similar socioeconomic and genetic backgrounds , but who did not meet our epidemiologic definition of resistance . hiv - infected sex - workers from the same cohort were also studied . assay optimization showed that the cm10 proteinchip array ( cation exchanger ) bound the greatest diversity of proteins from genital tract secretions and was chosen for subsequent analysis . a total of 579 cvl samples collected at multiple time points from 330 individuals were analyzed by seldi - tof ms . spectral analysis revealed a number of proteins that were differentially expressed in the hiv - resistant sex - worker group compared to controls . in particular , one 6 kda protein was over expressed in the genital tract secretions of the hiv - resistant study group compared to hiv - uninfected ( p & lt ; 0 . 001 ) and hiv - infected individuals ( p & lt ; 0 . 000001 ) ( fig1 ). this 6 kda protein was purified from cvl samples and identified using tandem ms as the 6 . 0 kda form of trappin - 2 , also known as human elafin / skalp ( skin - derived anti - leukoproteinase ) ( fig2 ). trappin - 2 is a member of the antileukoproteinase superfamily ( als ) of protease inhibitors . it has been largely studied in the context of lung disease , where its &# 39 ; antiprotease activity is thought to play a role in maintaining tissue integrity 10 . trappin - 2 has also been shown to have anti - bacterial and anti - inflammatory properties ( 10 , 11 ). the protein is produced at mucosal surfaces by epithelial cells and possibly macrophages in three major forms : a 12 . 3 kda cell associated form , as well as two secreted forms , the 9 . 9 kda pre - trappin - 2 and a 6 kda active form . trappin - 2 also shares partial homology with secretory leukocyte protease inhibitor ( slpi ), the other member of the als family , previously shown to inhibit the infection of macrophages by hiv ( 12 ). despite their homology , these two proteins exhibit significant structural and functional differences ( 10 ), as discussed above . an enzyme immunoassay for trappin - 2 was used to confirm the elevated levels of trappin - 2 in the genital tract of hiv - resistant women . as no specific antibody to the 6 . 0 kda form of the protein is available , an antibody specific for both the 6 . 0 and 9 . 9 kda forms of the protein and an antibody specific to the 9 . 9 kda form was used . over expression of trappin - 2 in the hiv - resistant study group compared to hiv - uninfected and hiv - infected individuals was confirmed ( p = 0 . 01 and p = 0 . 004 respectively , fig3 a ). interestingly , the 9 . 9 kda form of trappin - 2 was elevated in the hiv - resistant population compared to hiv - uninfected and hiv - infected individuals ( p = 0 . 002 and p = 0 . 01 respectively , fig3 b ). the ms intensity values of the 6 kda trappin - 2 peak correlated strongly with the values obtained via the elisa ( p = 0 . 000001 ), but showed no correlation with the 9 . 9 kda elisa data , illustrating the specificity of our ms approach . finally , since trappin - 2 has significant homology with slpi , we assessed slpi levels in genital tract secretions by elisa . no differences in slpi levels were found between study groups . multivariate analysis of trappin - 2 levels showed no relationship between levels of this protein with potential confounding variables such as menses , contraceptive use and concomitant infections . finally , the ability of trappin - 2 ( 6 . 0 kda form ) to inhibit hiv infection in an established hiv infection model was examined . a highly infectable t cell line ( mt4 ) was used to assay the effect of trappin - 2 inhibition on hiv hxbc2 replication 13 , 14 , 15 . hiv inhibition experiments were performed by preincubating trappin - 2 with either the virus , or target cells . a one - hour pre - exposure of cells to trappin - 2 at physiological concentrations significantly reduced virus replication ( fig4 ). these data suggest that hiv entry was being prevented , since the simultaneous addition of trappin - 2 and virus to target cells resulted in significantly less inhibition . when trappin - 2 was added back into the culture system after the initial one - hour preincubation , there was an even more dramatic decrease in viral replication ( fig4 b ). these data suggest that even a 2 - 3 fold increase in trappin - 2 concentration at mucosal surfaces , such as that noted in the hiv - resistant sex workers , could potentially have significant in vivo effects . in our in vitro system a 5 fold increase in trappin - 2 ( fig4 b ) had significant effects on hiv replication . trappin - 2 has previously been reported to have immunomodulatory effects and thus its influence on immune regulation is of interest . to determine if the hiv inhibitory effects of trappin - 2 are the result of induction of anti - viral cytokines such as ifn - γ , cytokine responses of mt4 cells after treatment with trappin - 2 were measured . no induction of cytokines was detected . although other inhibitors of hiv have been found at the mucosal barrier such as slpi and the chemokine sdf - 1 , they have not been shown to correlate with natural immunity to hiv 12 16 . recently , we described elevated levels of the β - chemokine rantes within the genital tract of hiv - resistant women 17 . here we have identified a new inhibitor of hiv within the genital tract of these women , using a powerful new technology . in conjunction with other naturally expressed inhibitors of hiv such as rantes , trappin - 2 may be forming an effective barrier within the genital tract of the hiv - resistant women , preventing infection . given the strong correlation of trappin - 2 with resistance , we believe that this protein plays an important role in preventing hiv infection . when we stratified women who had greater than the mean trappin - 2 levels ± 1 sd found in the hiv - uninfected group , we were able to show that individuals who had trappin - 2 levels greater than this amount showed a significantly reduced likelihood of being hiv infected , with a protective odds ratio ( or ) of 5 . 9 ( ci95 % 1 . 9 - 24 . 4 , p & lt ; 0 . 006 ) towards the hiv - resistant group based on the ms data ( only 4 / 30 hiv - uninfected , or susceptible women had trappin - 2 levels over this threshold , compared to 65 / 114 of the hiv - resistant women ), or an or of 4 . 5 ( ci95 % 1 . 24 - 24 . 7 , p & lt ; 0 . 002 ) based upon the elisa data ( 3 / 26 susceptible vs . 73 / 116 resistant ). of particular interest is the activity of trappin - 2 against an x4 type virus . this is unexpected , as current models of hiv pathogenesis suggests that r5 type viruses are responsible for the majority of sexual transmission ( 18 ). while elevated levels of chemokines binding ccr5 , such as rantes , may play a role in protection against sexual acquisition of hiv , the strong association of hiv - resistance to elevated trappin - 2 implies that ideal microbicides should be effective against both x4 and r5hiv viruses . clearly , further studies are needed to elucidate the anti - viral mechanism ( s ) by which trappin - 2 inhibits hiv , and its ability to inhibit across a variety of hiv strains and clades . however , the fact that resistant sex - workers demonstrate reduced susceptibility to multiple hiv clades in kenya , including a - d and recombinants , implies that this protein has broad activity . in conclusion , we have identified an innate immune protein , trappin - 2 , which is significantly elevated in the genital tract of hiv - resistant sex - workers , and has potent anti - hiv activity at physiologic concentrations . trappin - 2 may play an important role in mediating natural immunity to hiv infection and may have potential as an hiv microbicide . while the preferred embodiments of the invention have been described above , it will be recognized and understood that various modifications may be made therein , and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention . 1 . kaul , r . et al . hiv - 1 - specific mucosal cd8 + lymphocyte responses in the cervix of hiv - 1 - resistant prostitutes in nairobi . j immunol 164 , 1602 - 11 ( 2000 ). 2 . kaul , r . et al . hiv - 1 - specific mucosal iga in a cohort of hiv - 1 - resistant kenyan sex workers . aids 13 , 23 - 9 ( 1999 ). 3 . macdonald , k . s . et al . human leucocyte antigen supertypes and immune susceptibility to hiv - 1 , implications for vaccine design . immunol lett 79 , 151 - 7 ( 2001 ). 4 . quinn , t . c . & amp ; overbaugh , j . hiv / aids in women : an expanding epidemic . science 308 , 1582 - 3 ( 2005 ). 5 . fowke , k . r . et al . resistance to hiv - 1 infection among persistently seronegative prostitutes in nairobi , kenya . lancet 348 , 1347 - 51 ( 1996 ). 6 . devito , c . et al . mucosal and plasma iga from hiv - exposed seronegative individuals neutralize a primary hiv - 1 isolate . aids 14 , 1917 - 20 ( 2000 ). 7 . devito , c . et al . mucosal and plasma iga from hiv - 1 - exposed uninfected individuals inhibit hiv - 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human immunodeficiency virus 1 activity in vitro . j clin invest 96 , 456 - 64 ( 1995 ). 13 . harada , s ., koyanagi , y . & amp ; yamamoto , n . infection of htlv - iii / lav in htlv - 1 - carrying cells mt - 2 and mt - 4 and application in a plaque assay . science 229 , 563 - 6 ( 1985 ). 14 . ratner , l . et al . complete nucleotide sequence of the aids virus , htlv - iii . nature 313 , 277 - 84 ( 1985 ). 15 . lavallee , c . et al . requirement of the pr55gag precursor for incorporation of the vpr product into human immunodeficiency virus type i viral particles . j virol 68 , 1926 - 34 ( 1994 ). 16 . agace , w . w . et al . constitutive expression of stromal derived factor - 1 by mucosal epithelia and its role in hiv transmission and propagation . curr biol 10 , 325 - 8 ( 2000 ). 17 . iqbal , s . m . et al . elevated t cell counts and rantes expression in the genital mucosa of hiv - 1 - resistant kenyan commercial sex workers . j infect dis 192 , 728 - 38 ( 2005 ). 18 . meng , g . et al . primary intestinal epithelial cells selectively transfer r5hiv - 1 to ccr5 + cells . nat med 8 , 150 - 6 ( 2002 ). 19 . yao , x . j . et al . vpr stimulates viral expression and induces cell killing in human immunodeficiency virus type 1 - infected dividing jurkat t cells . j virol 72 , 4686 - 93 ( 1998 ).