6) 138–151www.elsevier.com/locate/yviro

Virology 355 (200

Viral evolution in macaques coinfected with CCR5- and CXCR4-tropicSHIVs in the presence or absence of vaccine-elicited anti-CCR5

SHIV neutralizing antibodies

Brian Burke a,b, Nina R. Derby a,b, Zane Kraft a, Cheryl J. Saunders a, Chuanbin Dai a,b,Nicholas Llewellyn c, Irina Zharkikh a, Lucia Vojtech a, Tuofu Zhu c, Indresh K. Srivastava d,

Susan W. Barnett d, Leonidas Stamatatos a,b,⁎

a Seattle Biomedical Research Institute, Seattle, WA 98109, USAb Department of Pathobiology, University of Washington, Seattle, WA 98195, USAc Department of Laboratory Medicine, University of Washington, WA 98195, USA

d Chiron Corporation, Emeryville, CA 94608-2916, USA

Received 28 March 2006; returned to author for revision 8 June 2006; accepted 11 July 2006Available online 22 August 2006

Abstract

Macaques were immunized with SF162 Env-based gp140 immunogens and challenged simultaneously with the CCR5-tropic homologousSHIVSF162P4 and the CXCR4-tropic heterologous SHIVSF33A viruses. Both mock-immunized and immunized animals became dually infected.Prior immunization preferentially reduced the viral replication of the homologous virus during primary infection but the relative replication of thetwo coinfecting viruses during chronic infection was unaffected by prior immunization, despite the fact that five of six immunized animalsmaintained a significantly lower overall viral replication that the control animals. Neutralizing antibodies participated in controlling thereplication of SHIVSF162P4, but not that of SHIVSF33A. Dual infection resulted in the emergence and predominance within the circulating CCR5virus pool, of a variant with a distinct neutralization phenotype. The signature of this variant was the presence of three amino acid changes ingp120, two of which were located in the receptor and coreceptor binding sites. Also, a significant fraction of the viruses circulating in the blood,as early as two weeks post-infection, was recombinants and prior immunization did not prevent their emergence. These findings provide newinsights into the dynamic interaction of CCR5- and CXCR4-tropic HIV isolates that are potentially relevant in better understanding HIV-mediatedpathogenesis.© 2006 Elsevier Inc. All rights reserved.

Keywords: SHIV; CCR5; CXCR4; HIV; Neutralizing antibodies; Viral recombination

Introduction

HIV envelope-based immunogens have so far been ineffec-tive in eliciting potent cross-reactive neutralizing antibodies(NAbs), in particular against heterologous primary HIV isolates(Bower et al., 2004; Earl et al., 2001; Gao et al., 2005; Garrity etal., 1997; Gzyl et al., 2004; Haigwood et al., 1992, 1990;Hanson, 1994; Kim et al., 2003; Mascola et al., 1996; VanCott

⁎ Corresponding author. Seattle Biomedical Research Institute, 307 WestlakeAvenue North, Seattle, WA 98109, USA. Fax: +1 206 256 7229.

E-mail address: leo.stamatatos@sbri.org (L. Stamatatos).

0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.virol.2006.07.026

et al., 1997; Yang et al., 2004a). Although recent improvementshave been made in the design of soluble HIV oligomericenvelope immunogens (gp140s) to elicit cross-reactive NAbs,the antibody responses they elicit are still weak in breadth andpotency (Barnett et al., 2001; Dong et al., 2003; Grundner et al.,2005; Yang et al., 2001, 2004b). Therefore, it is anticipated thatcurrently available HIV Env immunogens will fail to protect avaccinee from infection by isolates expressing envelopeglycoproteins that are heterologous to the immunogen. Evenmore alarming is the fact that it is very difficult, in the macaque/SIV or macaque/SHIV models, to even elicit by Env-basedimmunization protocols sterilizing immunity against viruses

139B. Burke et al. / Virology 355 (2006) 138–151

that express envelope glycoproteins that are homologous to theEnv immunogen (Amara et al., 2001, 2002b; Buckner et al.,2004; Davis et al., 2000; Doria-Rose et al., 2003; Earl et al.,2002; Fultz et al., 2003; Horton et al., 2002; Hu et al., 1992;Johnson et al., 2005; Lifson et al., 2004; Murphy et al., 2000;Ourmanov et al., 2000; Patterson et al., 2004, 2002; Polacinoet al., 1999a,b,c; Ramsburg et al., 2004; Robinson et al.,1999; Rose et al., 2001; Shiver et al., 2002; Willey et al.,2003; Zhao et al., 2003). However, priming by immunizationof anti-HIV Env-specific B cell responses does offer toimmunized macaques protection from disease developmentwhen the challenge virus expresses an envelope glycoproteinthat is homologous to the immunogen and, in general, theinclusion of the viral envelope glycoprotein in the vaccineformulation enhances the protective potential of the antiviralresponses elicited following infection (Amara et al., 2002a;Letvin et al., 2004).

All the above immunization/challenge animal studies wereconducted with a single virus as a challenge. In the present study,we examined how the replication of two viruses, one expressingan envelope glycoprotein that is homologous to the Envimmunogen and a second virus expressing an envelopeglycoprotein that is heterologous to the immunogen, will beaffected in a dually infected host, by the presence or absence ofpre-existing (vaccine-elicited) neutralizing antibody responsesthat target preferentially one of the two infecting viruses. To thisend, following immunization of macaques with HIV-1 SF162-derived Env gp140 immunogens, the animals were simulta-neously exposed to two SHIVs: SHIVSF162P4, expressing anenvelope that is homologous to our immunogens and SHIVSF33A

expressing an envelope that is heterologous to our immunogens.These viruses share a common SIVmac239 genetic backbone(they differ in Env by approximately 20%) and replicate to hightiters in macaques when inoculated individually (Harouse et al.,1998). Also, SHIVSF33A and a virus related to SHIVSF162P4,termed SHIVSF162P3, can both replicate to high titers in duallyinfected macaques (Harouse et al., 2003), in the absence of pre-existing anti-SHIV immunity.

Our results indicate that immunization with our SF162-derived gp140 Env constructs did not offer sterilizingimmunity during intravenous challenge, but significantlyreduced the early replication of the homologous, but not theheterologous, virus during acute infection. Immunization alsoresulted in a significant reduction in the overall plasmaviremia, in five of six macaques, during chronic infectionwithout affecting the relative replication of the two coinfectingviruses. Nor did immunization prevent the emergence ofrecombinant (within the env region) viruses. However, thepresence of the X4-tropic virus resulted in the emergence andpersistence of a R5 clone that is a minor species in theSHIVSF162P4 inoculum, indicating that during coinfection withR5- and X4-tropic viruses a complex and dynamic interactionbetween the two viruses takes place at the cellular level.Although our studies were conducted in the SHIV rhesusmacaque animal model, we believe that they are pertinent toinfection of humans by HIV and provide information that isrelevant to HIV-mediated pathogenesis.

Results

Development of antienvelope binding antibodies duringimmunization

During the DNA phase of immunization anti-Env antibodyresponses were detectable in only three animals, L170 andL667, in the ΔV2gp140 immunized group, and I658, in theGM154gp140 immunized group (Fig. 1A). As we and otherspreviously reported, the binding anti-Env antibody titers wereweak, transient and decreased to undetectable levels within2–3 months following the third DNA immunization (Buckneret al., 2004; Cherpelis et al., 2001). However, a boosting ofbinding anti-Env antibody titers was recorded 2–4 weeksfollowing immunization with the recombinant envelope gp140proteins. These titers decreased rapidly (by half or more)during the subsequent 2 weeks of observation. Animal H266,immunized with SF162gp140, had the lowest binding anti-Envantibody titers.

Neutralization potential of vaccine-elicited serum antibodies

(A)Homologous NAbs: During the DNA phase of immuniza-tion, anti-SHIVSF162P4 neutralizing antibody responses wereundetectable, as previously reported (Barnett et al., 2001;Buckner et al., 2004; Srivastava et al., 2003b). Followingrecombinant gp140 protein boost, all immunized animals(irrespective of the Env gp140 immunogen) developed potentbut comparable anti-SHIVSF162P4 NAb titers (Fig. 1B, top twopanels). Animal H266 had the lowest titers of NAbs, most likelybecause it developed the lowest titers of anti-HIV Env antibodiesin general (see above). The homologous neutralizing antibodytiters decreased in all animals (to varying degrees) over the next2weeks.We also evaluated the neutralizing potency of these seraagainst HIV-1 SF162 (Fig. 1B, bottom two panels). As expected(SHIVSF162P4 and HIV-1 SF162 express envelope glycoproteinsvery similar in amino acid composition) all animals elicitedpotent anti-HIV-1 SF162 NAbs. Differences in IC50 betweenHIV-1 SF162 and SHIVSF162P4 most likely are due to the factthat during neutralization, replication competent virus grown inPBMC was used in the case of SHIVSF162P4, while single roundcompetent virus generated by 293T cell transfection was used inthe case of HIV-1 SF162. (B) Heterologous neutralizingantibodies: we were also interested in examining whether thethree gp140 Env immunogens elicited NAbs against hetero-logous HIV-1 isolates. To this end, sera collected 2 weeksfollowing immunization with recombinant gp140 were evalu-ated against single round competent viruses expressing theenvelope glycoproteins of 33A, HxB2, 89.6, JRFL, ADA, andYU2 (Table 1). The two animals immunized with SF162gp140did not elicit anti-89.6 and anti-HxB2 Nabs. In contrast, allanimals immunized with the modified ΔV2gp140 orGM154gp140 immunogens elicited anti-89.6 and anti-HxB2NAbs. However, all other isolates tested here were resistant toneutralization. Therefore, these two envelopemodifications alterthe immunogenic properties of SF162gp140 proteins (as wepreviously described in the case of ΔV2gp140 (64)), but not in

Fig. 1. Antibody responses during immunization. (A) Relative end-point anti-Env antibody titers and (B) neutralizing antibody titers against SHIVSF162P4 (top twopanels) and HIV-1 SF162 (bottom two panels) were determined as described in the Materials and methods section. Neutralizing activity was evaluated at 2 and 4 weekspost-recombinant gp140 protein boosting.

140 B. Burke et al. / Virology 355 (2006) 138–151

ways that will result in the generation of NAbs against difficult toneutralize heterologous HIV-1 isolates.

Plasma viremia in macaques dually infected with SHIVSF162P4

and SHIVSF33A

We previously reported that in naïve rhesus macaques,SHIVSF162P4 replicates very efficiently, reaching acute plasmaviremia levels in the order of 107 to 108 viral RNA copies per ml(Buckner et al., 2004; Cherpelis et al., 2001). Similar levels ofreplication have been reported in SHIVSF33A-infected macaques(Harouse et al., 2003). In this study, the simultaneous inoculation

Table 1Neutralization of heterologous HIV-1 isolates by immune sera

Animal Immunogen Isolates

33A HxB2 89.6 JRFL ADA YU2 MuLV

H266 SF162gp140 (–) 10 (–) (–) (–) (–) (–)I375 SF162gp140 (–) (–) (–) (–) (–) (–) (–)I646 GM154gp140 (–) 30 10 (–) (–) (–) (–)I658 GM154gp140 (–) 100 10 (–) (–) (–) (–)L170 ΔV2gp140 (–) 30 30 (–) (–) (–) (–)L667 ΔV2gp140 (–) 30 15 (–) (–) (–) (–)

Neutralization was recorded as described in the Materials and methods sectionusing single round competent virions expressing the indicated envelopes. Thevalues indicate the serum dilution at which 50% neutralization was recordedwith sera collected 2 weeks following the recombinant envelope gp140immunization against sera collected prior to immunization. (–): 50%neutralization was not recorded at the lowest serum dilution tested (1:10).

of mock-immunized rhesus macaques with SHIVSF162P4 andSHIVSF33A (100 TCID50 each) resulted in peak plasma viremiaof 106–108 RNA copies/ml (Fig. 2A). No differences in acuteplasma viremia (day 14) levels were recorded between themock-immunized and immunized animals (P=0.22). Subsequently,the plasma viremia levels in the mock-immunized animalsdecreased and then fluctuated during chronic infection between2×104–105 RNA copies/ml. The plasma viremia in five of thesix immunized animals decreased to levels that were signifi-cantly lower (P=0.026, days 203–267) than those recorded inthe mock-immunized animals. In one immunized animal (I375)plasma viremia became undetectable. In the sixth immunizedanimal, H266, the levels of plasma viremia were higher than inthe mock-immunized animals and gradually increased toapproximately 108 RNA copies per ml. Such an unusuallyhigh level of viremia had not been previously observed inmacaques infected with SHIVSF33A or SHIVSF162P4 (or therelated SHIVSF162P3 virus) (Buckner et al., 2004; Harouse et al.,2003; Harouse et al., 1999). This animal developed AIDS andwas euthanized 239 days post-infection.

CD4+ T lymphocyte numbers during dual infection

Within 2 weeks after challenge, a reduction in the numbers ofperipheral CD4+ T lymphocytes occurred in both mock-immunized and immunized animals (Fig. 2B). This reductionis most likely due to infection with SHIVSF33A since the R5-tropic SHIVSF162P4 does not induce such a significant loss of

Fig. 2. Plasma viremia and CD4+ T lymphocyte numbers. (A) Plasma viremia was monitored by the b-DNA assay (dashed line indicates the limit of detection 125RNA copies per ml). (B) CD4+ T lymphocyte numbers per μl of blood were determined as described in the Materials and methods section.

141B. Burke et al. / Virology 355 (2006) 138–151

peripheral CD4+ T lymphocytes during acute infection(Buckner et al., 2004). The CD4+ T cell loss was sustained inthe two mock-immunized animals (M030 and R044) and in theSF162gp140-immunized animal H266 that did not controlplasma viremia (Fig. 2A). In the remaining immunized animalsa rebound of CD4+ Tcell numbers occurred. With the exceptionof immunized animal I646 (GM154gp140-immunized), thesenumbers remained stable throughout the period of observation.

The animals were euthanized at different times (day 203:L667, I646 and I658; day 239: H266; day 246: M030; day 407:R044, L170 and I375) during chronic infection in order todetermine the CD4+ T cell numbers in various lymph nodes(axillary, inguinal, mesenteric, bronchial, and iliac) as well asthe spleen. A severe CD4+ T lymphocyte depletion wasrecorded in all these tissues in all animals. CD4+ T lymphocyteswere either absent from these tissues or constituted less than 4%of all CD3+ T lymphocytes present. However, with theexception of animal H266 and I646, both of which experienceda sustained loss of peripheral CD4+ T lymphocytes, theimmunized animals in this study had not yet developed clinicalsigns of AIDS at the time of euthanasia.

Relative ratio of SHIVSF162P4, SHIVSF33A, and recombinantviruses in the blood

Viral RNA was isolated from plasma collected from eachmacaque during acute viremia (day 14 post-infection), and attwo time points during chronic infection, at day 99, and from theday the animal was euthanized (between 203 and 407 days post-infection). The C2V5 region of gp120 was amplified and thePCR fragments were cloned and sequenced or further amplifiedfor use in heteroduplex mobility assays (HMA) to determine therelative ratio of SHIVSF162P4 and SHIVSF33A viruses. At leasttwenty individual clones from each time point were examined.In addition, several of the clones that were analyzed by HMAwere also sequenced for confirmation.

Our results indicate that all animals were infected with bothSHIVSF162P4 and SHIVSF33A (Table 2). During acute infection(day 14), the two viruses replicated differently in the two mock-immunized animals. In animal M030, SHIVSF162P4 waspredominant (70%). In contrast, in animal R044, SHIVSF33A

was predominant (63%) at this time. These results are inagreement with those reported by others (Harouse et al., 2003)that these two R5- and X4-tropic SHIVs are equally efficient incoinfecting and coreplicating in macaques during intravenouslychallenge. Following clearance of acute viremia (day 99),SHIVSF162P4 predominated over SHIVSF33A in the blood of bothmock-immunized animals, consistent with previous reports ofR5- and X4-tropic dually challenged naïve animals (Harouse etal., 2003). The relative proportion of SHIVSF162P4 in the mock-immunized animals was 80% and 95% (M030 and R044animals, respectively) at that point.

Recombinant (in the env region) viruses were undetectablein the periphery of the mock-immunized animals during acuteinfection. Such recombinants were detectable however at 99days post-infection in animal M030 (15% of viral species).

In the immunized groups SHIVSF162P4 replication wassignificantly reduced (P=0.022) during acute infection (day 14),but not that of SHIVSF33A (P=0.37), as compared to themock-immunized macaques. Following acute infection, how-ever, at day 99, a predominance of SHIVSF162P4 was recordedin all the immunized animals, similar to what occurred in themock-immunized animals. At day 99, SHIVSF162P4 represented95% (I658), 60% (H266), 50% (I646), and 100% (I375, L170and L667) of all the viral species present in the periphery ofthe immunized animals. Although the relative proportion ofSHIVSF162P4 increased in the immunized animals betweendays 14 and 99, the absolute plasma viremia was 11- to 104-foldlower than during peak acute infection in these animals.Interestingly, recombinant viral species were present in five ofthe six immunized animals (representing 5–10% of the totalviral pool) as early as 14 days post-infection. At day 99 post-

Table 2Plasma viremia and relative ratio of circulating viruses during acute and chronic infection

Macaque DPI a CopiesRNA/ml

Percent of clones b Estimated Relative copies RNA/ml c

P4 33A Recom. P4 33A Recom.

M030 (Mock) 14 1.3×108 70 30 ND 9.1×107 3.9×107 –99 2.3×104 80 5 15 1.8×104 1200 3400246 1.5×105 53 11 37 8.0×104 1.6×104 5.6×104

R044 (Mock) 14 3.6×107 37 63 ND 1.3×107 2.3×107 –99 1.4×104 95 5 ND 1.3×104 700 –457 7.6×104 45 30 25 3.4×104 2.3×104 1.9×104

H266 (SF162) 14 3.5×107 10 85 5 3.5×106 3.0×107 1.8×106

99 5.3×105 60 ND 40 3.2×105 – 2.1×105

239 9.0×107 ND 12 88 – 1.1×107 7.9×107

I375 (SF162) 14 5.5×106 75 15 10 4.1×106 8.2×105 5.5×105

99 270 100 ND ND 270 – –457 130 58 42 ND 75 55 –

I646 (GM154) 14 6.4×106 5 85 10 3.2×105 5.4×106 6.4×105

99 9400 50 50 ND 4700 4700 –203 1600 78 22 ND 1250 350 –

I658 (GM154) 14 1.3×107 5 90 5 6.5×105 1.2×107 6.5×105

99 2200 95 5 ND 2100 100 –203 1100 50 20 30 550 220 330

L170 (ΔV2) 14 1.5×107 5 95 ND 7.5×105 1.4×107 –99 1.4×104 100 ND ND 1.4×104 – –457 7500 ND 84 16 – 6300 1200

L667 (ΔV2) 14 6.2×107 10 85 5 6.2×106 5.3×107 3.1×106

99 550 100 ND ND 550 – –203 1300 33 67 ND 430 870 –

ND or (–): not detected.a Days post-infection.b Determined by HMA and sequencing analysis.c Based on the total RNA copies per ml and the percent of SHIVSF162P4 (P4), SHIVSF33A (33A), or recombinant (Recom.) clones.

142 B. Burke et al. / Virology 355 (2006) 138–151

infection, however, recombinant species were detectable only inanimal H266 (40% of the total viral species).

At later times during chronic infection (days 203–407), amixture of viruses was present in the blood of all animals. Themock-immunized animals contained SHIVSF162P4, SHIVSF33A

and recombinant species, with SHIVSF162P4 being the pre-dominant virus (between 45% and 53% of the total viralspecies). Even though these percentages are lower than thoserecorded at day 99, the absolute number of SHIVSF162P4 viralparticles increased during that period (Table 2). Only oneimmunized animal, I658, had all three viral species present(SHIVSF162P4 50%, SHIVSF33A 20%, and recombinant 30%) atthese later stages of infection. In three other immunized animals(I375, L667, and I646) the two challenge viruses were detected(with SHIVSF162P4 present at 33–75% of the total viralpopulation) but not the recombinant virus. In the remainingtwo immunized macaques (L170 and H266) only SHIVSF33A

and recombinant virus were detectable, not SHIVSF162P4. L170contained 84% SHIVSF33A and 16% recombinant species, whileH266 contained a majority of recombinants (88%) withSHIVSF33A making up the rest of the viral species (12%).

Emergence of a specific SHIVSF162P4 variant

In mock-immunized macaques infected with onlySHIVSF162P4, envelope changes start to gradually emerge by70 days post-infection, concomitant with the appearance of

NAbs (Stamatatos, L., L.G. Gines, C.J. Saunders, L. Vojtech, I.Srivastava, S.W. Barnett, J.T. Safrit, C. Buckener, W. Blay, N.Haigwood, and R. McCaffrey Successful containment of viralreplication in macaques infected with an R5-tropic SHIVdepends on the early development of antienvelope antibodies.21st Annual Symposium on Nonhuman Primate Models forAIDS, 2003) (and data not shown). To examine whether theenvelope of SHIVSF162P4 evolved in a similar manner in thepresence of the competing SHIVSF33A virus, and also toexamine whether and how the envelope of SHIVSF33A evolvedin parallel, we performed longitudinal sequence analysis of theplasma virus envelope (C2V5). This envelope region of allSHIVSF33A viral clones sequenced throughout the course ofinfection was very similar to that found in the inoculatingSHIVSF33A viral stock swarm (data not shown). However, in thecase of SHIVSF162P4, in all animals, irrespective of whether theywere previously immunized or not, a specific envelope variantemerged shortly after infection and predominated thereafter.The signature of this envelope variant consisted of threemutations as compared to the consensus challenge SHIVSF162P4

C2V5 sequence (Fig. 3). These mutations were located in theC3 (S361P), V4 (Q385R) and C4 (Y425H) regions of gp120.This triple mutant (termed PRH) was detected as early as dayseven post-infection in the immunized animals L170 and I658(data not shown). By day 14, this triple variant was establishedin both the mock-immunized and immunized animals, with theexception of H266, irrespective of the relative ratio of the R5

Fig. 3. Emergence of the PRH mutant in the dually infected animals. The C2 to V5 region of the gp120 glycoprotein was sequenced longitudinally from all animals.The positions of the three amino acid changes: S to P, Q to R, and Y to H are indicated by shaded boxes. The number of clones containing this sequence out of the totalSHIVSF162P4 clones sequenced is shown on the right of each sequence. None: only SHIVSF33A (and/or recombinant) virus was detected.

143B. Burke et al. / Virology 355 (2006) 138–151

and X4 viruses (Fig. 3 and Table 2). At day 99 the PRH viruscomprised the majority of the SHIVSF162P4 species in allanimals. Subsequently, the PRH variant was detected in four ofthe six animals in which SHIVSF162P4 species were present. Inthe other two animals (R044 and I658) the consensus SHIV-SF162P4 virus was detectable, but not the PRH variant. Inmacaques H266 and L170 only the X4-tropic virus (andrecombinants, see below) was detectable at that time ofinfection. Interestingly, the PRH variant did not appear todevelop quasispecies character as very few clones includedadditional mutations in the C2V5 region (Fig. 3).

Phenotypic analysis of the PRH virus

The PRH variant is a minor species in the SHIVSF162P4

inoculum (approximately 3%) (data not shown). Its selection inSHIVSF162P4-infected macaques was never recorded previously.However, this variant was selected for in both the mock-immunized and immunized animals studied here. Since anti-Envantibody responses were not detectable in the mock-immunizedanimals during the first 10 weeks of infection (data not shown),the emergence of the PRH variant appears to be independent ofthe presence of anti-Env antibody responses and most likely aconsequence of the simultaneous infection by SHIVSF33A.

The S361P change is located in the CD4 binding site; theQ385R is in the outer domain of gp120; and the Y425H changeis close to residues involved in CCR5 recognition (Kwong et al.,1998). Specifically, S365 of HxBc2, which is analogous to S361in SF162, was shown to participate in backbone–backbonecontacts with CD4 (Kwong et al., 1998). An amino acid changefrom serine to proline could significantly alter these contactsand/or induce local conformational changes in this region of theCD4 binding site. Mutation of this analogous serine in NL4-3was shown to impair viral infectivity (Otto et al., 2003).Mutation of a tyrosine residue on HIV-1 YU2 gp120, that isanalogous to Y425 of SHIVSF162P4, reduced CCR5 binding(Rizzuto and Sodroski, 2000; Rizzuto et al., 1998). We weretherefore interested in determining whether the PRH variantdisplayed a different receptor/coreceptor interaction to that ofSHIVSF162P4, which is CD4 and CCR5 dependent (Harouse etal., 1999), offering a replication advantage to the PRH virus overSHIVSF162P4. We introduced these three mutations into thebackbone of the SF162 envelope gp160 to investigate thispossibility. Single round competent viruses expressing theSF162 Env with the PRH mutations were incubated with cellsexpressing CD4 and CCR5, CD4 and CXCR4, CCR5 alone, orCXCR4 alone and the extent of viral entry was monitored. Ourresults indicate that the PRH mutations do not affect Env

144 B. Burke et al. / Virology 355 (2006) 138–151

functionality (the SF162 Env containing the PRH mutationsmediates virus-cell fusion with the same efficiency as theparental SF162 envelope) and do not alter the utilization ofCD4, CCR5, and CXCR4 by the envelope (data not shown).

Development of neutralizing antibodies following infection

As mentioned above, anti-SHIVSF162P4 but not anti-SHIVSF33A NAbs were developed during immunization (Fig.1 and Table 1). We were interested to examine whether anti-SHIVSF33A NAbs developed following infection; whether priorimmunization with SF162 Env-derived immunogens affectedthe development of anti-SHIVSF33A NAbs; and whether theNAbs developed during infection neutralized the PRH variant.During these neutralization assays we used pseudovirusesexpressing the SF162 envelope glycoprotein, since HIV-1SF162 and SHIVSF162P4 express the same envelope and havevery similar neutralization profiles (Fig. 1B). A cloned enve-lope gp160 from SHIVSF33A (provided by Dr. C. Cheng-Mayer, ADARC) was used to generate single round competentvirions expressing the SHIVSF33A envelope. The PRHmutations were introduced into the SF162 gp160 envelopeglycoprotein.

Fig. 4. Neutralization susceptibility by serum antibodies developed during coinfectioneutralization susceptibility of SF162 (black symbols), PRH (red symbols) and 33A (banimals was determined as described in the Materials and methods section.

The mock-immunized animals did not develop potent anti-SHIVSF33A NAbs during the period of observation, but bothdeveloped high titers of anti-PRH NAbs (Fig. 4). Interestingly,although animal R044 developed strong anti-SF162 NAbs,animal M030 developed very weak anti-SF162 NAbs. Incontrast, all the immunized animals developed anti-PRH NAbswhile maintaining the production of anti-SF162 NAbs. Anti-SHIVSF33A NAbs were generated only in some immunizedanimals (H266, I375, L170, and I646) and only during the latestages of the observation period. Anti-SHIVSF33A NAbs wereundetectable in the immunized animals at 99 days post-infection. This suggests that NAbs had very little to do withthe extent of replication of SHIVSF33A during the first 99 days ofinfection in these dually infected animals. Interestingly, thePRH variant was several fold more susceptible to neutralizationthan SF162 to neutralization by serum antibodies collected at allstages of infection.

Discussion

Previously, we showed that immunization of macaques withgp140 Env constructs derived from the HIV-1 SF162 envelopeglycoprotein resulted in the long-term control of viral

n. The PRH mutations were introduced on the SF162 Env background and thelue symbols) to serum antibodies developed longitudinally in the dually infected

Fig. 4 (continued).

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replication of a SHIV virus, SHIVSF162P4, which expresses anenvelope that is homologous to the immunogens used (Buckneret al., 2004). In this study, we wished to determine if therewould be similar control of viral replication when macaquesimmunized with SF162 envelope-based gp140 immunogens,were simultaneously exposed to two viruses: one (SHIVSF162P4)that expresses an envelope homologous to the immunogens andanother virus (SHIVSF33A) that expresses an envelope which isheterologous to the immunogens. To this end, we immunizedmacaques with three different SF162-derived gp140 envelopeconstructs (SF162gp140, ΔV2gp140, and GM154gp140) priorto challenge with SHIVSF162P4 and SHIVSF33A and thenmonitored longitudinally viral replication and evolution.

All three immunogens elicited potent homologous NAbs butonly the modified immunogens (ΔV2gp140 and GM154gp140)elicited heterologous NAbs. However, these cross-reactiveneutralizing antibody responses were weak and very narrowin breadth, targeting viruses that are considered to be easilyneutralizable. NAbs against the heterologous SHIVSF33A viruswere undetectable during immunization with the three gp140immunogens evaluated here.

Following viral challenge the immunized animals hadindistinguishable peak plasma viral loads from the mock-immunized animals. Similarly, no significant difference in theRNA copies of SHIVSF33Awas detected at this time between the

mock-immunized and immunized animals. However, the earlyreduction of the homologous SHIVSF162P4 virus in theimmunized animals suggests that the immune responses elicitedduring immunization contributed to the initial control of thisvirus, in accordance to our previous observations made duringsingle infection with SHIVSF162P4 (Buckner et al., 2004).

Following acute viremia (by day 99), five of the siximmunized macaques had lower overall viral loads than themock-immunized animals. At that point, the SHIVSF162P4 viruspredominated in the two mock-immunized and in 5/6immunized animals. The predominance of SHIVSF162P4 overSHIVSF33A following acute infection is consistent to previousreports (Harouse et al., 2003) and appears to be unrelated to theimmune responses elicited during immunization. The predomi-nance of the R5-tropic SHIVSF162P4 virus at this time could inpart be due to the reduction of CD4+ T lymphocytes from theperiphery, that occurred in all animals within 2 weeks post-infection. Peripheral CD4+ T lymphocytes are the major targetsfor the X4-tropic SHIVSF33A virus, while the R5-tropicSHIVSF162P4 virus preferentially replicates in the GALTassociated CD4+ T lymphocytes (Harouse et al., 1999). Withthe exception of animal H266, the immunized animals were ableto control viral replication (see Table 2) to levels that weresignificantly lower than those recorded in the mock-immunizedanimals during chronic infection. Additionally, the relative

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replication of the two viruses during chronic infection differedfrom animal to animal most likely due to the fact that these areout bread nature animals.

A consequence of dual or super infection is the emergence ofrecombinant viruses (Esteves et al., 2003; Grisson et al., 2004;Gundlach et al., 2000; Sabino et al., 1994). Recombinant viralspecies (within the C2V5 envelope region) were present in bothmock and immunized animals. In certain cases (animals L667,I646, and I375) recombinants were present early but not late ininfection. In other animals (M030, R044, and L170) recombi-nant viral species appeared later during infection. Theseobservations are indicative of a dynamic viral replication, andpotential competition, of the various viral species present inthese animals, even though these viruses display distinctcoreceptor usage. The Env recombinant viruses we identifiedhere may not accurately represent the total recombinant viralspecies present in these animals since recombination could haveoccurred outside the env region of the viral genome. Theidentification of such recombinants is not feasible sinceSHIVSF162P4 and SHIVSF33A share the same genome outsidethe env.

Interestingly, although anti-SHIVSF162P4 NAbs developedearly in the coinfected animals, anti-SHIVSF33A NAbs werenot developed until much later during infection, and only in afew animals. Therefore, although NAbs most likely wereinvolved in regulating the replication of SHIVSF162P4, they didnot participate in regulating the replicative potential ofSHIVSF33A. We can speculate at this point that the reductionin SHIVSF33A replication following primary infection was dueto either a reduction in available target cells, and/or to theearly emergence of anti-SHIVSF33A specific T cell responses.The involvement of cell-mediated immunity in the relativecontrol of replication of the two coinfecting viruses meritsdetailed investigation.

Since SHIVSF162P4 and SHIVSF33A utilize different cor-eceptors and preferentially replicate in different anatomicalsites, why do recombinant viruses emerge so early afterinfection? The coreceptor utilization of these viruses wasevaluated in vitro using established cell lines as target cells(Harouse et al., 1999). These in vitro results may notaccurately predict what occurs in vivo. It is also conceivablethat in vivo, individual cells expressing both CCR5 andCXCR4 become infected by the two viruses. It is also possiblethat cells, some of which are infected with SHIVSF33A andsome by SHIVSF162P4, form syncytia. These possibilities couldlead to the emergence of recombinant viruses.

After infection a single viral clone (PRH) predominated theSHIVSF162P4 viral pool in both the mock-immunized andimmunized animals. As this variant is detected early (7–14days post-infection) in seven of eight macaques, including themock-immunized animals, it is unlikely to be the result ofantibody responses, as these responses were not detected untilmuch later in the mock-immunized animals (data not shown).Because the PRH variant does not predominate duringSHIVSF162P4 single challenge studies, the emergence and thepredominance of the PRH virus in the dually infected animalsappears to be related to the presence of the coinfecting

SHIVSF33A virus. Additionally, the PRH variant is highlysusceptible to neutralization by antibodies elicited during thisdual infection. It is difficult to find a rational explanation forwhy a neutralization susceptible variant, such as PRH, wouldemerge during infection, unless it has a replicative advantageover the other viruses that circulate in these animals. So, it ispossible that PRH emerged as a result of competition at thecellular level with the SHIVSF33A virus. The PRH mutationsare located at the C3, V4, and C4 regions of gp120. Themutations in C3 (S361P) and C4 (Y425H) are located inregions of the viral envelope glycoprotein that interact withCD4 and possibly influence CCR5 binding, respectively(Kuroda et al., 1998; Rizzuto and Sodroski, 2000). However,the PRH mutations do not alter the CD4 and CCR5dependence of the SF162 envelope. One caveat here is thatthe PRH mutations were examined in the context of the SF162Env. It is therefore possible that on the actual virus thatreplicated in those animals, other mutations located outside theC2V5 region, in combination with the PRH mutations,conferred to the envelope a receptor/coreceptor recognitionphenotype that is distinct from that of SF162 Env or of theSF162 Env with the PRH mutations. We did sequence theentire gp120 and part of the extracellular region of gp41 fromthree PRH clones from one animal and we did not record anyother major amino acid changes (data not shown). However,sequencing and analysis of additional PRH full-length gp160sequences are necessary to examine this possibility in moredetail. An additional possibility is that the PRH mutationsallow the virus to utilize coreceptor molecules other thanCCR5 (here we only examined the possibility that PRH canutilize CXCR4 or not). This is something that also needsfurther exploration. It would be interesting to test whether asimilar emergence of the PRH variant would occur when thecoinfecting virus is different than SHIVSF33A, for example,SHIV89.6P (which also utilizes CXCR4) or SIVmac239(which utilizes primarily CCR5). A previous study hasindicated a complex relationship in the relative replicativeproperties of SHIV89.6P and SIVmac251 in dually infectedmacaques (Wolinsky et al., 2004). In that study theexperimental reduction in the replication of the R5-tropicvirus resulted in a parallel increase in the replication of the X4virus. However, in that study the potential emergence ofspecific viral variants was not examined.

It could be argued that the experimental situation presentedhere (i.e., coinfection by R5- and X4-tropic viruses) will notoccur frequently during natural HIV-1 infection. R5-tropicHIV-1 viruses are those that predominantly establish infectionin naïve individuals. However, there is evidence of transmis-sion of X4-tropic HIV-1 viruses (Cornelissen et al., 1995;Groenink et al., 1991; Roos et al., 1992; Schuitemaker et al.,1992; van't Wout et al., 1994) and there is evidence thatmultiple variants can be transmitted simultaneously (Ritola etal., 2004). The primary aim of this study was to examine howimmunization with our current gp140 Env immunogens wouldaffect the relative replication of coinfecting viruses. Based onour results, we would anticipate that extensive recombinationbetween the infecting HIV-1 viruses will occur very soon after

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infection, especially when these variants utilize the samecoreceptor molecules, and that immunization will not alter thefrequency of recombination. However, our immunogenselicited NAbs against one of the two infecting viruses and itwould be interesting to examine whether similar observationswill be made if the immunogens elicit neutralizing antibodyresponses against both viruses.

In summary, our studies indicate that until immunogens andimmunization strategies are designed that would elicitsterilizing immunity against HIV, establishment of infectionby diverse isolates would be possible and recombinationamong these isolates would occur early. Potentially, suchrecombinants could affect the rate of progression to diseaseand be transmissible, in which case future vaccines mustprevent their spread. Our studies also indicate that a complexinteraction may occur during HIV infection between CCR5-tropic and CXCR4-tropic viruses and this interaction mayaffect both the development of antiviral immune responses aswell as pathogenesis.

Materials and methods

Animals, immunogens and immunization protocol

Adult rhesus macaques of Indian origin were housed at theTulane National Primate Research Center. The immunogensused in these studies were derived from the SF162 envelope.Three related immunogens were used, SF162gp140,ΔV2gp140and GM154gp140, which were generated by introducing stopcodon sequences upstream from the transmembrane region ofthe SF162 (Stamatatos and Cheng-Mayer, 1998), SF162ΔV2(Stamatatos and Cheng-Mayer, 1998) and GM154 envelopes(Ly and Stamatatos, 2000), respectively. SF162ΔV2 wasderived from SF162, by deleting 30 out of the 40 amino acidsof the second hypervariable regions (V2 loop). GM154 was alsoderived from SF162 by eliminating an N-linked glycosylationsite at the amino terminal side of the V2 loop (position 154)which protects SF162 from antibody-mediated neutralization(Ly and Stamatatos, 2000). DNA vectors expressing these threeimmunogens were generated as we previously described(Barnett et al., 2001).

The animals received three DNA immunizations at 4-weekintervals as we previously described in detail (Barnett et al.,2001). Following a resting period of 40 weeks, the animalsreceived a fourth DNA immunization and at the same timethey were immunized with the corresponding purifiedoligomeric gp140 protein, emulsified with the MF-59Cadjuvant. A total of 2 mg DNA in 1 ml of endotoxin-freewater was administered each time per animal. 1.6 mg wasadministered intramuscularly at the quadriceps and 0.4 mgwere administered intradermally at two sites. A total of 0.1 mgof protein in 0.5 ml total volume was administeredintramuscularly to the deltoids of each animal. Mock-immunized animals received the ‘empty’ DNA vector andthe MF-59C adjuvant. The DNA vectors expressed the threegp140 proteins with intact gp120-gp41 cleavage sites. Incontrast, this site was eliminated by mutagenesis from the

corresponding recombinant gp140 proteins (Srivastava et al.,2003a).

Viral challenge

Four weeks following the last immunization, the animalswere challenged intravenously with 100 TCID50 of cell-freeSHIVSF162P4 virus (Harouse et al., 1999) and 100 TCID50 ofcell-free SHIVSF33A (Harouse et al., 1998). The SHIVSF162P4

and SHIVSF33A viruses were provided by Drs. Cheng-Mayerand Harouse (ADARC) and were propagated in Dr. Stamata-tos's laboratory once in PHA-activated human peripheral bloodmononuclear cells (PBMC) prior to administration to themacaques. Following challenge, blood was collected twiceduring the first week, on a weekly basis for the subsequent 3weeks and monthly thereafter. The animals were euthanized atdifferent times during the course of infection (in order todetermine viremia and CD4+ T cell loss at various tissues) orwhen they developed AIDS. PBMC and plasma were isolatedfrom each bleed and from the time of death, and were keptfrozen for further analysis.

Determination of CD4+ T cell numbers

CD4+ T lymphocytes in the blood were determined with theuse of an anti-CD4 antibody coupled to PE (clone MT477,Becton Dickinson). The number of CD3+ T lymphocytes wasdetermined with the use of an anti-CD3 antibody coupled toFITC (clone SP34, Becton Dickinson).

Plasma viral load determination

Plasma viremia (RNA copies per ml of blood) wasdetermined at the Bayer Reference Testing Laboratory (Berkley,CA). The lower limit of detection is 125 RNA copies per ml.

Determination of viral genotype

To determine the ratio of SHIVSF162P4 and SHIVSF33A

virions circulating in the blood of dually challenged animals, weemployed the heteroduplex mobility assay (HMA) (Delwart etal., 1993; Harouse et al., 2003). Briefly, viral RNAwas isolatedfrom plasma of infected animals, using the QIAamp viral RNAmini kit (QIAGEN, Valencia, CA), DNAase-treated, andreverse transcribed using oligo (dT)20 and SuperScript IIIreverse transcriptase (Invitrogen, Carlsbad, CA). Transcriptswere amplified from the cDNA by PCR with the primers P5(binds to the C2 region) (Zhu et al., 1993) and PE2 (binds to theC5 region) (Zhu et al., 2002) using Platinum Taq (Invitrogen).These primers amplify the SHIVSF162P4 and SHIVSF33A

envelopes with equal efficiency. PCR reactions were carriedout on 2 μl of cDNA using 4 mM Mg2+, 800 nM dNTPs, and100 nM of each primer. The PCR reaction consisted ofdenaturation at 95 °C for 4 min; 25–45 cycles of 95 °C 15 s,55 °C 30 s, and 72 °C 45 s; and a final extension of 10 min at72 °C (Fulcher et al., 2004; Zhu et al., 2002). Amplifiedproducts were gel purified using the QIAquick gel extraction kit

148 B. Burke et al. / Virology 355 (2006) 138–151

(QIAGEN), ligated into TOPO TA cloning vector pCR 2.1(Invitrogen) and transformed into DH5α-T1 competent cells(Invitrogen). An additional round of PCR was carried out fromindividual clones (up to twenty from each time point) and theproduct purified as above. Each sample was heat denatured inannealing buffer (100 mM NaCl, 10 mM Tris–HCl, pH 7.8, and2 mM EDTA) at 95 °C for 3 min in the presence of either a ‘P4probe’ or a ‘33A probe’ and annealed on wet ice. These ‘probes’correspond to the C2V5 region of SF162 (‘P4 probe’) orSHIVSF33A (‘33A probe’) envelope gp160. The samples werethen run on 5% polyacrylamide gel at 250 V for 2 h, visualizedby ethidium bromide staining, and the numbers of P4-like and33A-like clones present at each time point were determined.Clones that were detected with both the ‘P4 probe’ and ‘33Aprobe’ were defined as recombinants (in the C2V5 region)between the two viruses.

Analysis of sequence diversity

The nucleotide sequences of the above HMA samples orother C2V5 transcripts amplified by PCR using the primersED7 (5′-CTGTTAAATGG CAGTCTAGC) and ED8 (5′-CACTTCTCCAATTGTCCCTCA) were sequenced using theprimers PE2 (binds to the V5 region of gp120) and M13F (5′-CAGGAAACAGCTA TGAC-3′) (binds to the TOPO TAcloning vector pCR 2.1 at the 5′ of the inserted HIV envelopesequence), respectively. Sequences were analyzed and alignedusing the EditSeq, MegAlign, and SeqMan sequence analysisprograms (DNASTAR).

Generation of mutant envelope glycoproteins

Introduction of the PRH mutations (a S361P in C3, a Q385Rin V4 and a Y425H in C4 regions of gp120) into the plasmidexpressing SF162 envelope gp160 (pEMC* SF162gp160)(McCaffrey et al., 2004; Saunders et al., 2005) was donesequentially using three primer sets via PCR and using theStratagene Quickchange Mutagenesis Kit according to themanufacturers protocol.

Determination of anti-HIV envelope binding antibodies

The relative titers of serum antibodies specific for the HIVenvelope were determined longitudinally during immunizationand following SHIV challenge as previously described(Buckner et al., 2004; Cherpelis et al., 2001). Briefly, solubletrimeric SF162gp140 (Chiron Corporation, Emeryville, CA)(0.5 μg/ml in 100 mM NaHCO3, pH 8.5) was used to coatImmulon 2HB 96-well plates (ThermoLabsystems, Franklin,MA) (0.1 ml per well) by an overnight incubation at RT.Following washing with TBS (25 mM Tris/144 mM NaCl, pH:7.6) the wells were blocked with SuperBlock (SB) (Pierce,Rockford, IL). Serially diluted (in SB with 0.05% Tween 20)heat-inactivated (56 °C for 35 min) sera were added to the wells(0.1 ml per well) for 1 h at 37 °C. Serially diluted pre-immunization sera from the corresponding animals were used asnegative controls. Unbound antibodies were removed by TBS-

washing and the HIV envelope-bound antibodies were detectedwith the use of a goat–antihuman IgG coupled to alkalinephosphatase (Zymed Immunochemicals, South San Francisco,CA) (1:30,000 dilution in SB and 0.05% Tween 20) (1-hincubation at 37 °C). The wells were washed with TBS andsubstrate for alkaline phosphatase (DAKO Cytomation, Car-pinteria, CA) was added (50 μl per well) for 1 h at roomtemperature. Upon addition of an equal amount of Amplifier(DAKO Cytomation, Carpinteria, CA) the OD490nm signalwas recorded from each well on a Vmax microplate reader(Molecular Devises, Sunnyvale, CA). A plot of the OD490 nmsignals versus serum dilution was generated and end-pointantibody titers were determined as the highest post-immuniza-tion (or post-viral challenge) serum dilution that produces anOD490 nm signal three times that of the OD490nm produced bythe pre-immunization sera at their lowest dilution.

Cells

The U87 human astroglioma cells expressing CD4 andCCR5 or CXCR4 were cultured as previously described(Saunders et al., 2005). TZM-bl cells expressing CD4, CCR5,and CXCR4 (Wei et al., 2003) and containing an integratedreporter gene for firefly luciferase under control of an HIV-1LTR were maintained in DMEM supplemented with 10% FBS,penicillin (100 U/ml), streptomycin (100 μg/ml), and L-glutamine (2 mM). Human embryonic kidney (HEK-293T)cells were maintained in the same growth media as the TZM-blcells.

Generation of single-round competent reporter viruses

Preparation of luciferase reporter viruses expressing HIVenvelope glycoproteins derived from SF162, PRH, 89.6, 33A,HxB2, JRFL, YU2, and ADA and capable of a single round ofreplication was carried out as previously described (Saunderset al., 2005).

Neutralization assays

Sera collected from the animals prior to the initiation ofthe immunization protocol (pre-bleeds), following the immu-nization with recombinant gp140 envelope glycoproteins, andat various points following infection, were heat inactivated(1 h at 56 °C), serially diluted in cell medium and incubatedfor 1 h at 37 °C with virus. The virion–serum mixture wasthen added to TZM-bl cells for 72 h. The supernatant wasremoved and the cells were then lysed with 0.1 ml 1× celllysis buffer (Cell Culture Lysis Reagent buffer, Promega) andfrozen at −80 °C for 2 h. Following thawing at roomtemperature, 0.06 ml from each well was transferred to aBlack Enhanced Binding 96-well solid plate, luciferasesubstrate (Promega) was added to each well (0.1 ml perwell) and the relative light units (RLU) associated with eachwell were recorded with a Fluoroskan Ascent FL (Thermo-Labsystems). The percent neutralization was calculated asfollows: (RLU in the wells containing pre-bleeds−RLU in

149B. Burke et al. / Virology 355 (2006) 138–151

wells containing post-immunization or post-infection sera /RLU in the wells containing pre-bleeds)×100. Single roundcompetent virus expressing the MuLV envelope was used as acontrol for non-specific serum anti-HIV neutralizing activity.Neutralization of SHIVSF162P4 was tested using a replicationcompetent virus grown in human PBMC.

Statistical analysis

Total plasma viremia, as well as individual plasma virallevels of SHIVSF162P4 and SHIVSF33A viruses, during primaryand chronic infection between mock-immunized and immu-nized animals was compared using t-test analysis. Throughoutthe present study, two-tailed P values are cited. A result isconsidered significant if the P value is <0.05 (5% level ofsignificance).

Acknowledgments

These studies were supported by NIH grants AI47708 andAI51217 to LS, and AI49109 and AI 55336 to T.Z., and theM. J. Murdock Charitable Trust and the J. B. PendletonCharitable Trust. HIV vaccine work at Chiron is supported byNIAID-NIH contracts and grants (AI-95367; I-AI-05396 and5PO1 AI48225-03). We thank Haiying Zhu for assisting withthe initial set up of the HMA experiments and Courtney Gradyfor technical assistance.

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