Characterization of anatomically compartmentalized plasma and milk simian immunodeficiency

48 Pages·2016·1.12 MB·English

JVI Accepted Manuscript Posted Online 27 July 2016
J. Virol. doi:10.1128/JVI.00701-16
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1 Characterization of anatomically compartmentalized plasma and milk simian
2 immunodeficiency virus variants in chronically infected African green monkeys.
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4 Jonathon E. Himes1, Carrie Ho1, Quang Nguyen1, Joshua D. Amos1, Haolin Xu2, Cliburn Chan2,
5 Shein-Chung Chow2, Christina Ochsenbauer6, Zhanna Kaidarova7, Sheila M. Keating7,
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6 Genevieve G. Fouda1, and Sallie R. Permar1,3,4,5,# w
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8 1Duke Human Vaccine Institute, and Departments of 2Biostatistics and Bioinformatics, o
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9 3Pediatrics, 4Immunology, 5Molecular Genetics and Microbiology, Duke University Medical t
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10 Center, Durham, NC. 6Department of Medicine, University of Alabama at Birmingham, //jv
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11 Birmingham, AL. 7Blood Systems Research Institute, San Francisco, CA. s
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13 Running Title A
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14 Plasma/milk SIV variants in chronically infected AGMs (54 characters) ,
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16 # Correspondence should be addressed to Sallie R. Permar ([email protected]) e
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18 (Abstract, 236 words; Manuscript, 6,945 words)
19 Keywords
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20 SIV, natural hosts, AGM, milk, vertical transmission
21 Abbreviations
22 AGM, African green monkey; env, envelope; HIV-1, human immunodeficiency virus type 1;
23 SIV, simian immunodeficiency virus; RM, rhesus monkey
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24 Abstract (236 words)
25 Unlike human immunodeficiency virus (HIV-1) infected humans, African-origin, natural simian
26 immunodeficiency virus (SIV) hosts, such as African green monkeys (AGMs), sustain
27 nonpathogenic SIV infections and rarely vertically transmit SIV to their infants. Interestingly,
28 chronically SIV-infected AGMs have anatomically compartmentalized SIV variants in plasma
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29 and milk, whereas humans and SIV-infected rhesus monkeys (RMs), Asian-origin non-natural o
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30 SIV hosts, do not exhibit this compartmentalization. Thus, it is possible that AGM SIV lo
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31 populations in milk have unique phenotypic features that contribute to the low postnatal e
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32 transmission rates observed in this natural host species. In this study, we explored this o
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33 possibility by characterizing the infectivity, tropism, and neutralization susceptibility of plasma t
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34 and milk SIVsab env variants isolated from chronically SIVsab92018ivTF-infected AGMs. //jv
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35 AGM plasma and milk SIVsab env pseudovirus variants exhibited similar infectivity, s
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36 neutralization susceptibility to autologous and heterologous plasma, and chemokine coreceoptor o
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37 usage for cell entry, suggesting similar ability to initiate infection in a new host. We also o
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38 assessed the cytokine milieu in SIV-infected AGM milk and compared it to that of SIV-infected p
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39 RMs. MIP-1β, G-CSF, IL-12/23, and IL-13 trended significantly higher in SIV-infected AGM 4,

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40 milk compared to that of RMs, while IL-18 and IL-6 trended significantly higher in SIV-infected 1
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41 RM milk compared to that of AGMs. Taken together, our findings imply that non-viral maternal y

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42 factors, such as the cytokine milieu, rather than unique characteristics of SIV populations in the e
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43 milk, contribute to the low postnatal transmission rates observed in AGMs.
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44 Importance (150 words)
45 Due to the ongoing global incidence of pediatric HIV-1 infections, including many that occur via
46 breastfeeding, development of effective vaccine strategies capable of preventing vertical HIV
47 transmission through breastfeeding remains an important goal. Unlike HIV-1-infected humans,
48 the natural SIV host species African green monkeys (AGMs) sustain nonpathogenic SIV
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49 infections, rarely transmit the virus postnatally to their infants, and exhibit anatomically o
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50 compartmentalized SIV populations in milk and plasma. Identifying unique features of the lo
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51 anatomically compartmentalized milk SIV populations could enhance our understanding of how e
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52 AGMs may have evolved to avoid transmission through breastfeeding. While this study o
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53 identified limited phenotypic distinctions between AGM plasma and milk SIV populations, t
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54 potential differences in milk cytokine profiles of natural and non-natural SIV hosts were //jv
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55 observed. These findings imply the potential importance of non-viral factors in natural SIV host s
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56 species, such as innate SIV/HIV immune factors in milk, as a means of naturally preventing o
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57 vertical transmission. o
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58 Introduction (479 words)
59 Now four decades into the HIV-1 epidemic, more than 200,000 pediatric human
60 immunodeficiency virus type 1 (HIV-1) infections continue to occur annually despite
61 considerable gains in antiretroviral-based prevention strategies (1, 2). Nearly half of these
62 ongoing pediatric transmission events occur postnatally via breastfeeding. The ARV-based
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63 approach to prevention of postnatal transmission falls short of preventing all infant infections o
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64 due to challenges such as adherence, access, viral resistance, and acute maternal infection during lo
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65 lactation. Thus, preventing this route of vertical HIV-1 transmission through an efficient e
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66 pediatric vaccine regimen remains essential for achieving a HIV-free generation. o
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67 Unlike HIV-1-infected humans, natural simian immunodeficiency virus (SIV) host t
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68 species such as African green monkeys (AGMs) sustain non-pathogenic SIV infection and rarely //jv
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69 transmit SIV vertically to infants (3-7), likely a result of >30,000 years of host-pathogen s
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70 coevolution (8). This lack of pathogenic infection and vertical transmission are not due to o
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71 complete control of viremia, as chronic viral replication populates both the systemic and breast o
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72 milk compartments in natural hosts (9-13). Several findings suggest that both maternal and p
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73 infant immunologic features contribute to the rare postnatal transmission rates in natural SIV 4,

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74 hosts. We previously demonstrated that lactating, SIV-infected AGMs exhibit less B cell 1
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75 dysfunction and more functional antibody responses in both plasma and milk compared to rhesus y

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76 monkeys (RMs), a non-natural SIV host with similar vertical transmission rates to HIV-infected e
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77 humans (12, 14). Moreover, the development of a neutralizing antibody response can arise early
78 after acute infection in AGMs (15). This neutralizing response within the mammary
79 compartment could contribute to the rare vertical transmission rates in breastfeeding AGMs.
80 Alternatively, studies have also demonstrated that infant AGMs (16) and sooty mangabeys (17),
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81 another natural SIV host species, exhibit low CCR5 coreceptor expression on CD4+ target cells,
82 which could decrease the infant’s risk of SIV acquisition.
83 In addition to characterizing maternal and infant immunologic features unique to natural
84 SIV host species, genotypic analyses of the viral populations in plasma and milk demonstrated
85 that, unlike in HIV-1-infected humans (18) and SIV-infected RMs (19), SIV-infected AGMs
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86 exhibit anatomically compartmentalized SIV variant populations in the milk and plasma (9). o
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87 While systemic SIV variants of natural hosts can be highly infectious and resistant to lo
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88 neutralization (20), breast milk SIV variants have not previously been characterized. Thus, the e
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89 potential existence of a phenotypically distinct milk SIV population could contribute to the rarity o
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90 of postnatal transmission in AGMs. In this study, we sought to characterize the infectivity, t
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91 neutralization, and tropism phenotype of milk and plasma SIV sabaeus (SIVsab) variants from //jv
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92 chronically infected AGMs in an attempt to elucidate unique features of these genotypically s
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93 distinct viral populations, which could contribute to genetic selection of SIVsab variants in the o
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94 milk compartment, or the low vertical transmission rates observed in AGMs. Identifying key o
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95 features of nontransmitted viral populations to which an infant is chronically exposed may p
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96 inform pediatric HIV-1 vaccine strategies to eliminate vertical transmission through 4,

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97 breastfeeding. 1
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99 Methods (2,552 words)
100 Study animals and specimen collection.
101 Six female AGMs and 4 female RMs were hormonally induced into lactation, followed by
102 intravenous inoculation with SIVsab92018ivTF or SIVmac251, respectively, as described
103 previously (14, 21). Blood and milk were collected at acute and chronic time points up to 2
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104 years post infection. Peripheral blood mononuclear cells (PBMCs) and plasma were isolated by o
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105 density gradient centrifugation using Ficoll-Plaque (GE Healthcare, Waukesha, WI). Milk of 5 lo
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106 uninfected, hormonally-induced lactating AGMs and 6 additional hormonally-induced lactating e
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107 SIVsab9351BR-infected AGMs (22) as well as 2 naturally lactating and 10 hormonally-induced o
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108 lactating, uninfected RMs, and 1 additional chronically SIVmac251-infected, naturally lactating t
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109 RM was employed to characterize the cytokine milieu in chronically-infected and uninfected //jv
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110 AGMs and RMs (21). Animals were maintained according to the Guide for the Care and Use of s
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111 Laboratory Animals (23). o
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112 o
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113 SIVsab env variant selection. p
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114 SIVsab plasma and milk env variants from 5 AGMs at 5 months post SIVsab92018ivTF 4,

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115 infection were amplified and sequenced using single genome amplification (SGA), as previously 1
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116 reported (9), and were found to be anatomically compartmentalized in 4 of 5 AGMs. For this y

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117 study, isolates from the 4 AGMs with statistically significant genetic compartmentalization of e
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118 milk and plasma virus variants were selected for plasma and milk env cloning and pseudovirus
119 production. Successfully cloned and functional SIVsab env pseudoviruses included 13 plasma
120 and 10 milk variants. These envs were amplified directly from the initial SGA product using
121 PCR and cloned into the pcDNA3.1D Topo mammalian expression vector (Invitrogen, Waltham,
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122 MA) using standard molecular biology techniques. Cloning was considered successful if the
123 ligated env sequence did not include non-synonymous mutations when compared to the SGA
124 sequencing results used to initially assess viral variant compartmentalization (9). In MEGA 6
125 (24), SIVsab variants were aligned by codon using MUSCLE (25) and Maximum-likelihood
126 trees were constructed using the general time-reversible substitution model with fixed gamma-
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127 distributed rate variation across sites. o
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128 lo
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129 Env pseudovirus variant production. e
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130 SIVsab env variant clones were co-transfected in 293T cells using Jetprime transfection reagent o
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131 (Polyplus Transfection, New York, NY) according to the manufacturer’s instruction. Both t
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132 SG3Δenv (26, 27) and NL4.3_lucΔenv backbones (supplied by David Montefiori; Duke //jv
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133 University) were employed in the co-transfection to produce functional, non-replication s
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134 competent pseudoviruses, as previously described (28). After a 48-hour incubation at 37°C, o
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135 culture supernatant was collected, sterile filtered, and stored at -80°C. SIVsab variant o
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136 pseudovirus functionality was determined by assessing infectivity as the 50% tissue culture p
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137 infectious dose (TCID50) in TZM-bl reporter cells. Other viruses used as controls throughout the 4,

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138 study and prepared in the same manner included pseudoviruses Du156.12 env, Mn.3 env, 89.6 1
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139 env, and SIVmac251.30 env, as well as the replication competent viruses SIVsab92018ivTF y

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140 (HQ378594.1), SIVmac239 IMC (M33262), and pNL-LucR.T2A-SIVagm.sab92018.ps. e
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142 Replication-competent SIV env encoding LucR reporter viruses
143 For certain experiments, replication competent reporter viruses encoding SIV env and Renilla
144 luciferase (LucR) were utilized. pNL-LucR.T2A-SIVagm.sab92018.ps was constructed by
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145 modifying the previously described pNL-LucR.T2A-Env.ecto HIV-1 reporter virus approach
146 (29, 30) such that SIV env genes of interest could be expressed in an established robust, isogenic
147 reporter virus backbone. It has been previously reported that SIV Env cytoplasmic tail
148 truncations to 22 or 23 aa are selected for by replication of SIVmac in human cells, including
149 PBMCs (31). We took advantage of this by only inserting the SIV env coding region
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150 encompassing the entire ectodomain, membrane spanning domain, and the first 22 aa of the o
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151 cytoplasmic tail into the NL-LucR.T2A proviral backbone; by introducing the premature stop lo
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152 codon at this position, the SIV sequence ends right before the SIV splice acceptor site for exon 2 e
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153 of tat and rev. This region also contains the SIV RRE, which has been found to work o
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154 sufficiently well in concert with HIV-1 Rev (32). In our pSP72mss HIV-1 env cloning t
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155 intermediate which contains a silent mutation BstBI restriction site in the membrane spanning //jv
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156 domain coding region (nt 8301-8306) (29), we mutated the HIV env start codon and 2 additional s
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157 methionine codons up to the KpnI site at NL4-3 nucleotide 6343 (located just 3’ of the end of the o
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158 vpu/env orf overlap) to create pSP72mssMet-minus. We PCR amplified the env region of interest o
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159 from SIVagm.sab92018 TF (HQ378594.1), introducing a 5’ KpnI and a 3’ BstBI site, removing p
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160 an internal KpnI site and introducing the premature env stopcodon (“ps”, at position 4,

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161 corresponding to SIVsab92018ivTF Env aa 728). The respective final PCR fragments were 1
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162 ligated into pSP72mssMet-minus via KpnI/BstBI. This essentially replaced the HIV env y

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163 sequence between the overlapping vpu orf and the splice acceptor sites for exon 2 of tat/rev with e
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164 that of SIVagm.sab92018.ps. The HIV/SIV chimeric region was ligated back into the NL-
165 LucR.T2A-mssD proviral backbone via the EcoRI and BamHI sites in HIV NL4-3, to generate
166 the SIV.env-IMC-LucR reporter proviral plasmid, pNL-LucR.T2A-SIVagm.sab92018.ps. Virus
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167 stocks were prepared by transfection of individual proviral DNA into 293T cells and titered on
168 TZM-bl cells essentially as described above.
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170 Infectivity measurements.
171 SIVsab env/SG3Δenv variant infectivity in TZM-bl cells was used to estimate viral infectivity.
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172 TCID50 of viruses was assessed in 96-well cell culture plates using Tat-regulated firefly o
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173 luciferase (LucF) reporter gene expression to quantify viral entry into various cell lines. Four lo
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174 replicate infectivity assays of each virus were performed and the Reed Muench method was used e
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175 to calculate TCID50 as previously described (28). Viruses were serially diluted onto cells and om

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176 allowed to infect for 48hr at 37°C prior to luminescence quantification (Promega, Madison, WI). t
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177 The minimum detection limit for infection was defined as 2.5 the average signal of the cell-only //jv
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178 controls. Prior to comparing plasma and milk SIVsab variant infectivity, TCID50 was s
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179 normalized to SIV Gag p24 concentration quantitated by ELISA (Perkin Elmer, Waltham, MA) o
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180 as an estimate of total viral particles to account for variations intrinsic to the transfection o
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181 protocol. To additionally assess viral infectivity in cells without CCR5 overexpression such as p
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182 that seen in TZM-bl cells, SIVsab92018ivTF infectivity of M7-luc cells (supplied by David 4,

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183 Montefiori; Duke University) was also measured (33) and compared to SIVsabivTF infectivity of 1
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184 TZM-bl cells. Variations of the human glioma cell line NP-2/CD4 with specific chemokine y

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185 coreceptor expression of CCR5, CXCR4, or GPR15, or no chemokine receptor (provided by Dr. e
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186 Feng Gao) (34), as well as HOS cell-derived Ghost(3) cells expressing CD4, CCR5, and CXCR4
187 (obtained from the NIH AIDS Reagent Program) (35) were used to assess
188 SIVsab/NL4.3_lucΔenv pseudovirus infectivity in the setting of these specific chemokine
189 receptors. NP-2 and Ghost(3) cell line chemokine receptor expression was verified through the
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