key: cord-0720460-xmwno35r
authors: Cele, Sandile; Karim, Farina; Lustig, Gila; San, James Emmanuel; Hermanus, Tandile; Tegally, Houriiyah; Snyman, Jumari; Moyo-Gwete, Thandeka; Wilkinson, Eduan; Bernstein, Mallory; Khan, Khadija; Hwa, Shi-Hsia; Tilles, Sasha W.; Singh, Lavanya; Giandhari, Jennifer; Mthabela, Ntombifuthi; Mazibuko, Matilda; Ganga, Yashica; Gosnell, Bernadett I.; Abdool Karim, Salim S.; Hanekom, Willem; Van Voorhis, Wesley C.; Ndung’u, Thumbi; Lessells, Richard J.; Moore, Penny L.; Moosa, Mahomed-Yunus S.; de Oliveira, Tulio; Sigal, Alex
title: SARS-CoV-2 prolonged infection during advanced HIV disease evolves extensive immune escape
date: 2022-01-14
journal: Cell Host Microbe
DOI: 10.1016/j.chom.2022.01.005
sha: 983e4bbfbda7631da2b707202450f2e3ec17be92
doc_id: 720460
cord_uid: xmwno35r

properties of variants coming from these regions. We mapped neutralization of a SARS-CoV-2 strain that evolved over 6 months from ancestral virus in a person with advanced HIV disease in South Africa, infected prior to emergence of the Beta and Delta variants. We longitudinally tracked the evolved virus and tested it against self-plasma and convalescent plasma from ancestral, Beta, and Delta infections. Early virus was similar to ancestral but evolved a multitude of mutations found in Omicron and other variants. It showed substantial but incomplete Pfizer BNT162b2 escape, weak neutralization by self-plasma, and despite pre-dating Delta, extensive escape of Delta infection-elicited neutralization. This example is consistent with the notion SARS-CoV-2 evolving in individual immune-compromised hosts, including those with advanced HIV disease, may gain immune escape of vaccines and enhanced escape of Delta immunity, with implications for vaccine breakthrough and reinfections.

We next characterized SARS-CoV-2 that had evolved in a person with advanced HIV diagnosed 140 in late September 2020 with SARS-CoV-2 infected with the ancestral lineage B.1.1.273 which we 141 previously described in a case report (Karim et al., 2021b) . The study participant was discharged 142 9 days post-diagnosis according to South Africa guidelines and remained asymptomatic for most 143 study visits (see description in the Materials and methods). HIV viremia persisted up to day 190 144 post-diagnosis due to irregular ART adherence. SARS-CoV-2 titer was high throughout this 145 period, ranging from a Ct of 16 to 27 (Karim et al., 2021b) . Last positive qPCR result was early 146 May 2021. Phylogenetic mapping is consistent with a single infection event (Fig 1B) . The CD4 147 count was <10 at enrollment (Fig 2A top row) . It increased at later timepoints, possibly due to the 148 improved adherence to ART and a switch to dolutegravir based therapy which reduced the HIV 149 viral load to below the level of clinical detection (Karim et al., 2021b) . SARS-CoV-2 was detected 150 by qPCR until day 216 post-diagnosis (Fig 2A second row) . We attempted to isolate live virus up 151 to and including the day 216 post-diagnosis swab sample. While there was insufficient sample to 152 isolate virus from the day 0 swab, we isolated and expanded SARS-CoV-2 from subsequent 153 swabs until and including day 190 post-SARS-CoV-2 diagnosis (Fig 2A third row) . Successful 154 isolation indicates that live virus was shed at that time. RBD specific IgG antibodies in the blood 155 were at borderline detection levels (slightly above the mean negative control + 2 std) at the early 156 timepoints but were detected at higher levels starting day 190 (Fig 2A fourth row, Fig S1 related  157 to Fig 2A) . 158 159 Outgrown virus was sequenced to detect majority and minority variants (Fig 2B) . The mutations 160 found in the outgrown virus were representative of the virus in the swab from the matched time 161 point (Table S2 related during expansion in Vero E6 cells and likely confers moderate neutralization escape (Johnson et 165 al., 2021) . E484K was first detected in the day 6 isolate (Fig 2B) . This mutation persisted at day 166 20 and 34 but was replaced with the F490S substitution starting on day 71 when the K417T 167 mutation was also detected. The N501Y mutation was detected in the virus isolated on day 190 168 post-diagnosis. Mutations were clustered in the RBD, including K417T, F490S, and N501Y in the 169 day 190 viral isolate ( Fig 2C) . Among the RBD mutations in the day 190 isolate, K417T is found 170 in the Gamma variant, and F490S is found in the Lambda variant. Among NTD mutations, T95I 171 is found in Mu, and R190K is at the same location as the R190S in Gamma. N501Y is found in 172

Beta, among others. The Omicron variant has emerged as this work was being revised and has 173 J o u r n a l P r e -p r o o f mutations at many of the same sites as the evolving virus described here 174 (https://covdb.stanford.edu/page/mutation-viewer/#sec_b-1-351). This includes the D796Y 175 mutation only found in Omicron among the major variants ( Fig 2B) . 176 177 We tested three of the isolates for neutralization: viruses outgrown from day 6 and day 20 swabs 178 (designated D6, D20) representing viruses from early infection, and virus outgrown from the day 179 190 swab (D190), after substantial evolution. Neutralization of the D6, D20, and D190 isolates by 180 self-plasma was low at the early timepoints ( Fig 2D) . However, neutralization of D6 and D20 was 181 evident by plasma sampled from day 190 and was more pronounced in the plasma sampled from 182 day 216. The D6 isolate was the most sensitive to neutralization by day 216 plasma. Neutralization 183 declined for D20 and further for D190, suggesting sequential evolution of escape ( Fig 2D) . The 184 ancestral virus and Beta and Delta variants were also tested for neutralization using day 216 185 plasma. Neutralization was lower for all three non-self viral strains relative to self-derived virus. 186

The strongest neutralization was of ancestral virus. Delta was neutralized to a lesser degree, and 187

Beta was not detectably neutralized ( Fig 2D) . 188

We also tested the D6, D20, and D190 isolates against plasma from other convalescent 190 participants infected with ancestral virus. Neutralization of D190 by ancestral infection elicited 191 plasma was decreased dramatically relative to D6, with FRNT50 for D190 being 9.3-fold lower 192 despite the presence of the E484K mutation in D6 ( Figure 2E ). The difference was smaller 193 between D190 and D20 (5.1-fold, Figure 2F ), consistent with evolution of some neutralization 194 escape in D20 relative to D6. We also tested neutralization of D190 virus using Pfizer BNT162b2 195 vaccinated participants. BNT162b2-elicited plasma neutralization capacity was decreased 5-fold 196 against D190 relative to ancestral virus with the D614G mutation ( Fig 2G) . We compared 197 neutralization of Beta, D6, D20, and D190 on a subset of remaining BNT162b2 plasma samples 198 from 5 participants 5-6 months post-vaccine, where neutralization declined to relatively low levels. 199

Despite this limitation, neutralization was detectable and showed a pattern consistent with the 200 other results: D190 neutralization escape was very similar to Beta, while D6 and D20 showed no 201 escape from BNT162b2 elicited neutralization (Fig S2 related to Fig 2G) . A 5-fold reduction is less 202 than the fold-drop we obtained for the Beta variant with convalescent plasma from previous 203 infection (Cele et al., 2021a) and these results are consistent with substantial but incomplete 204 escape of D190 from BNT162b2 elicited immunity. 205 206 J o u r n a l P r e -p r o o f We next assessed the serological distance between D190, D6, the ancestral strain, Beta, Delta, 207

and Alpha variants. We tested against convalescent plasma obtained from participants infected 208 with ancestral strains or Beta or Delta variants. Neutralization by ancestral plasma immunity 209 declined 8.8-fold relative to ancestral virus for the D190 isolate (Fig 2H) , similar to the Beta variant. 210

In contrast, there was only a 1.6-fold decline for the D6 isolate ( Fig 2I) . D190 virus was neutralized 211 relatively well by Beta variant infection elicited plasma, with a 2.6-fold reduction (Fig 2J) . This was 212 similar to D6 neutralization, which was reduce 2.3-fold relative to Beta (Fig 2K) . A much more 213 dramatic decline was observed with Delta variant elicited immunity: a 27.1-fold drop in 214 neutralization capacity compared to neutralization of Delta virus (Fig 2L) . Escape from Delta 215 elicited immunity was much more moderate for D6, with a 5.4-fold decline compared to Delta virus 216 ( Fig 2M) . 217

Mapping the results ( Fig 2N) shows that the Beta and Delta variants are serologically far apart, 219 with ancestral virus forming a hub. The greater distance of Beta relative to Delta from ancestral 220 is consistent with Beta being an escape variant and so evolving antibody escape mutations in the 221 RBD as well as mutations and deletions in the NTD. The Alpha variant was serologically like the 222 ancestral strain and was well neutralized by Beta plasma. However, it did escape Delta elicited 223 neutralization. D6 was serologically similar to the ancestral strain even though it had the E484K 224 substitution (and the in vitro evolved R682W which is reported to confer a moderate decrease in 225 neutralization). In contrast, the D190 virus, which underwent extensive evolution, was 226 serologically like Beta despite sequence differences. It had escape from ancestral and a much 227 more pronounced escape from Delta elicited plasma, similar to the Beta virus. We did not map 228

Gamma, but neutralization escape of the Delta but not the Beta variant from Gamma elicited 229 neutralization (Liu et al., 2021) Antigenic cartography has been extensively used in influenza (Smith et al., 2004) . Genetic 258 distance may be partially but not completely a proxy for antigenic distance, as some substitutions 259 can lead to large changes in antigenicity while others lead to minor effects (Smith et al., 2004) . It 260 is unclear whether genetic distance is a good measure for antigenic differences between SARS-261

CoV-2 variants. For example, based on the phylogenetic tree ( Fig 1B) it is not obvious that Beta 262 elicited immunity would neutralize Alpha virus better than Delta immunity. Perhaps this is because 263 SARS-CoV-2 evolution seems be closer to HIV, where multiple strains radiate from a common 264 ancestor (Korber et al., 2000) , than to the stepwise progression in influenza (Smith et al., 2004) . Characteristics of convalescent participants with ancestral, Beta, or Delta variant infection whose 371 blood was used for neutralization experiments can be found in Table S1 , and Pfizer BNT162b2 372 vaccinated blood donor characteristics can be found in Table S4 . 

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The mutations in day 190 isolated virus (D190) shown as red spheres. (D) Neutralization of day 893 6 isolated (D6), day 20 isolated (D20), and D190 virus by self-plasma collected days 6 to 216 and 894 the ancestral D614G, Beta and Delta viruses with plasma collected day 216

D6 (E) and D20 (F) relative to D190 virus by ancestral infection elicited plasma (n=8)

Neutralization of D190 compared to D614G by Pfizer BNT162b2 plasma (n=12). (H-I) 897

Neutralization of D190 (H) and D6 (I) compared to D614G by ancestral plasma (n=8)

Neutralization of D190 (J) and D6 (K) compared to Beta virus by Beta plasma (n=9)

Neutralization of D190 (L) and D6 (M) compared to Delta virus by Delta plasma (n=10)

GMT between virus strain on left and right. p-values are * 902

as determined by the Wilcoxon rank sum test

Summary map (not to scale) of serological distances as measured by fold-decrease in 904 neutralization. For clarity, Beta plasma neutralization of D6 is not shown

substantially but incompletely escaped BNT162b2-elicited immunity as well as neutralization by 8 self-plasma and showed extensive escape from neutralization elicited by Delta infections. 9 10Highlights 11 12• An ancestral SARS-CoV-2 infection persisting over 6 months evolved neutralization 13 escape 14 15• Evolved virus serological relationship is mapped to ancestral virus and key variants 16 17• Evolved virus shows greatest escape from Delta and least from Beta-elicited immunity 18 19• Evolved virus substantially but incompletely escapes BNT162b2 vaccine-elicited  20  antibodies  21  22  23  24  25  26  27