JCM Accepted Manuscript Posted Online 19 June 2019

J. Clin. Microbiol. doi:10.1128/JCM.00757-19

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

1 Decreased seroreactivity in individuals initiating antiretroviral therapy during acute HIV infection.

2 Manak Mark M1, Linda L Jagodzinski2, Ashley Shutt1, Jennifer A. Malia2, Mike Leos3, Jason
3 Ouellette1, Siriwat Akapirat4, Donn L Colby5, Nittaya Phanuphak5, Leigh Anne Eller1, Merlin L.
4 Robb1, Mark de Souza5, Jintanat Ananworanich1,5, and Sheila A Peel2, on behalf of the
5 RV254/SEARCH010 and the RV217 Study Teams.

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1
6 U.S. Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military
7 Medicine, Bethesda, MD USA; 2U.S. Military HIV Research Program, Walter Reed Army Institute of
8 Research, Silver Spring, MD USA; 3Bio-Rad Laboratories, Inc., Benicia, CA, USA; 4Department of
9 Retrovirology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; 5SEARCH,
10 Thai Red Cross AIDS Research Centre, Bangkok, Thailand.

11 J Clin Micro – Abstract

12 Antiretroviral therapy (ART) during acute HIV infection (AHI) interrupts viral dynamics and may
13 delay emergence of serological markers targeted by current HIV screening and confirmatory assays,
14 thus creating challenges for correctly classifying HIV infection status. The performance of three HIV
15 Ag/Ab Combo assays (Bio-Rad GS, Abbott Architect, and Bio-Rad BioPlex 2200) was evaluated on
16 samples collected from RV254/SEARCH010 (Bangkok, Thailand) participants at week 12 and 24
17 following initiation of ART at Fiebig I (FI) (N=23), FII (N=39), or FIII/IV (N=22). Supplemental,
18 confirmatory testing was performed by Geenius HIV 1/2 and HIV-1 Western Blot (Bio-Rad). Samples
19 from 30 untreated, HIV-1 infected individuals demonstrated robust HIV Ag/Ab Combo reactivity with
20 well-developed HIV-1 Western Blot profiles by 24 weeks after infection. In contrast, 52.2% of
21 samples from individuals initiating ART at FI, 7.7% at FII, and 4.5% at FIII/IV were non-reactive by
22 HIV Combo assays, with 36.4-39.1% of samples had low (<10) Signal to Cut Off (S/CO) results on
23 Architect and BioPlex assays. Seroreversion from reactive to non-reactive status was observed in 10
24 individuals initiating ART at FII, and 3 at FIII/IV. Geenius and HIV-1 Western Blot results were
25 negative or indeterminate for 73.9% and 69.6% of individuals treated at FI; 50.0% and 26.3% at FII;
26 and 54.5% and 40.9% at FIII/IV, respectively. Virologic suppression of HIV-1 by ART during AHI
27 impedes seroconversion to biomarkers of infection, limiting the utility of HIV Ag/Ab Combo and
28 supplemental, confirmatory assays for infection status determination.

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29 Key words: Human Immunodeficiency Virus, HIV Ag/Ab Combo assay, HIV Geenius assay,
30 Seroreversion, Diagnostics, Antiretroviral Therapy, Acute HIV Infection

31 Corresponding Author
32
33 Mark Manak, Ph.D.
34 Department of Diagnostics and Monitoring
35 Walter Reed Army Institute of Research

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36 503 Robert Grant Ave., Room 1A32
37 Silver Spring, MD 20910
38 Phone: 301 319-3512
39 FAX: 301 319-9997
40 Email: mmanak@hivresearch.org
41

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42 Background
43 Current guidelines recommend initiation of antiretroviral therapy (ART) as soon as possible after HIV
44 diagnosis to minimize risk of forward transmission, interrupt seeding of latent reservoirs, delay disease
45 progression, and ultimately enhance overall health outcomes (1-7). However, reduction or elimination
46 of viral expansion by ART in acute HIV infection (AHI) to below the threshold required for evolution
47 of HIV immune response may block or delay the emergence of HIV-specific antigen (Ag) and/or

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48 antibody (Ab) targets for detection of HIV infection (8-11). Negative results on standard HIV Ab tests
49 are common among HIV-1 infected children who initiate ART at an early age (12-15). At two years of
50 age, about half of all HIV-1 infected infants who initiated ART within 2 weeks of birth were
51 seronegative by HIV immunoassays and rapid device tests, compared to 11% who were seronegative if
52 ART was initiated at 12 and 24 weeks of age. All children initiating ART later than 24 weeks of age
53 were seropositive by HIV immunoassays at two years of age (6, 16). In the absence of ongoing
54 antigenic stimulation, seroconversion on second-generation immunoassay Ab tests also failed to occur
55 among adult participants treated during AHI (9, 15, 17, 18). Several studies reported seroreversion
56 from reactive to non-reactive results by HIV Ag/Ab Combo (Combo) assays in HIV infected
57 participants who initiated ART during AHI (8, 14, 15, 17-20). Delay in the appearance of serological
58 markers, and in some cases reversion to non-reactive results was observed in participants who initiated
59 ART during AHI prior to the development of full Ab response (RV254/South East Asia Research
60 Collaboration in HIV (SEARCH) 010 Study, Thai Red Cross AIDS Research Centre, Bangkok,
61 Thailand) (18). The present study expands upon the aforementioned RV254/SEARCH010 study and
62 assesses the ability of current HIV screening and supplemental, confirmatory assays to detect HIV
63 biomarkers of infection in this early treated cohort.

64

65 Materials and Methods

66 Clinical Specimens. Plasma from HIV infected participants used in this evaluation were selected from
67 two studies in which very early HIV infection was identified by screening of individuals at high risk of
68 HIV infection with the Hologic Aptima HIV-1 RNA Qualitative Assay (Aptima). The evolution of
69 serological markers in 30 untreated participants was examined in samples obtained from the RV217
70 Early Cohort HIV Study conducted in Kenya, Uganda, Tanzania and Thailand (21, 22) and was

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71 compared to those of participants enrolled in the RV254/SEARCH010 Study in Bangkok, Thailand,
72 who initiated ART prior to establishment of fully evolved HIV-1 Ab responses (10, 13, 18, 23). The
73 stage of HIV infection at time of ART initiation was assigned based on the Fiebig staging system:
74 Fiebig I (HIV RNA+, p24 Ag-, HIV Ab-; N=23), Fiebig II (HIV RNA+, p24 Ag+, HIV Ab-; N=39);
75 Fiebig III (HIV Ab+, WB-; N=13); and Fiebig IV (HIV Ab+, HIV-1 WB indeterminate; N=9) (18, 24).

76 Assays. Serial specimens from untreated individuals and from those who initiated therapy at Fiebig I-

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77 IV of infection were tested at the HIV Diagnostic Reference Laboratory (HDRL) in Silver Spring, MD
78 by the Bio-Rad GS HIV Combo Ag/Ab Combo (BRC) EIA, Bio-Rad GS HIV-1/HIV-2 PLUS O EIA
79 (1/2/O), Bio-Rad GS HIV-1 Western Blot (HIV-1 WB), and GeeniusTM HIV 1/2 (Geenius)
80 Supplemental assays. The Bio-Rad Genscreen HIV-1 Ag Assay (Ag) was used to determine the
81 concentration of serum HIV-1 p24 antigen. Testing of untreated individuals (RV217 cohort) was
82 conducted at frequent intervals for up to 4-7 weeks after first Aptima reactivity by 1/2/O and BRC
83 assays. HIV-1 WB testing for 15 of 30 individuals was conducted on serial samples from first Aptima
84 reactive sample to 24 weeks. Individuals treated at Fiebig I-IV (RV254 cohort) were tested by 1/2/O,
85 BRC, WB and Geenius assays at weeks 1, 2, 12 and 24. Abbott Architect HIV Ag/Ab combo (ARC)
86 testing and assignment of Fiebig stages of RV254/SEARCH010 samples was performed at the Thai
87 Red Cross AIDS Research Centre, Bangkok, Thailand as previously described (18). Bio-Rad BioPlex®
88 2200 HIV Ag-Ab Combo (BPX) testing was performed at Bio-Rad, Inc in Benicia, CA.

89 Statistical Analysis. Calculations of correlations, linear regression and paired t-test parametric analysis
90 were conducted in Prism 7 (GraphPad Software, La Jolla, CA).

91 Regulatory Approval Clinical specimens used in this study were collected between 2009-2014 under
92 Human Subjects Research protocols approved by the Institutional Review Boards at the respective
93 collecting institutions and at the Walter Reed Army Institute of Research (WRAIR). Specimens were
94 provided blinded to the laboratory with no accompanying private health or personal identifying
95 information provided. The investigator(s) adhered to the policies regarding the protection of human
96 subjects as prescribed by Code of Federal Regulations (CFR) Title 45, Volume 1, Part 46; Title 32,
97 Chapter 1, Part 219; and Title 21, Chapter 1, Part 50 (Protection of Human Subjects) and Army
98 Regulation 70-25 (25)(26)(27).

99

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100 Results
101 The time course of evolution of serological markers of HIV-1 infection in untreated individuals was
102 evaluated on serial samples collected from participants in the RV217 study who were identified in
103 AHI. Figure 1A shows the results of the BRC assay for 30 individuals whose first Aptima reactive
104 sample was detected within 3-4 days of last non-reactive result. BRC yielded a reactive result on
105 average at 6.5 +/-2.8 days (range 2-11 days) after first detection of RNA and rose rapidly to saturated

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106 Signal to Cut Off (S/CO) value of the assay (S/CO: 12-16) by 3-5 weeks and remained high thereafter.
107 The evolution of serological reactivity from time of first RNA detection is shown in Figure 1B. The
108 timing of earliest BRC reactivity corresponds to that of p24 Ag assay results and represents reactivity
109 to the HIV-1 p24 Ag component of the BRC assay. The 1/2/O assay which detects only IgM/IgG Ab
110 was reactive on average by day 15.0 +/-4.8 (range 6-22) after first Aptima RNA reactive sample. Both
111 the 1/2/O and BRC assays reached maximum assay S/CO of 10-13 and 12-16, respectively, by week 2-
112 3 and remained high throughout subsequent testing. HIV-1 WB positivity is shown by percent of
113 participants with a positive result. All participants were HIV-1 WB positive by 4-5 weeks and
114 remained positive at subsequent time points, as expected.

115 Evolution of p24 antigenicity and HIV-1 seroreactivity after ART initiation for each individual
116 (RV254) was evaluated by p24 Ag, BPX, and ARC assays. All samples from FII-IV stage individuals
117 prior to ART initiation contained quantifiable p24 Ag by the Bio-Rad Ag Assay and detectable p24 Ag
118 by BPX but were p24 Ag non-detectable within one week of therapy and remained p24 Ag non-
119 detectable at week 12 and 24 on ART (not shown). Figure 2 shows the evolution of Ag/Ab combo
120 results by BPX (solid lines), or by ARC (dotted lines) in cases where insufficient sample was available
121 to test by BPX prior to treatment initiation. For those cases where both assays were performed on the
122 same sample, the BPX and ARC assays yielded similar results. Samples from all 23 FI individuals
123 were initially non-reactive by Ag/Ab combo assays at week 0; 5 (21.7%) were weakly reactive (<10)
124 and two (8.8%) were strongly reactive (S/CO >10) by BPX or ARC assays by week 1. Samples from
125 12 FI individuals (52.2%) remained non-reactive throughout the 24-week period. All FII individuals
126 were Ag/Ab combo reactive at ART initiation (week 0), including 15 of 39 (38.5%) individuals with
127 strong BPX S/CO values, which declined after one week of therapy. Samples from several individuals
128 showed a transient increase in S/CO at week 1, followed by a subsequent decline. Seroreversion from
129 Ag/Ab combo reactive to non-reactive was observed in 10 of 39 (25.6%) individuals who initiated

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130 therapy at Fiebig II and 3 of 22 (15.6%) individuals at FIII/IV. By week 24, samples from 3 (7.7%)
131 FII individuals remained non-reactive, 15 (38.5%) were weakly reactive and 21 (53.8%) were strongly
132 reactive. Samples from individuals who initiated ART at FIII/IV, also showed an initial decline
133 (BPX), with samples from one (4.5%) individual remaining non-reactive, 8 (36.4%) weakly reactive,
134 and 13 (59.1%) strongly reactive after 24 weeks of treatment.

135 The percentage of samples from individuals grouped as non-reactive (<1.0), weakly reactive (<10), or

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136 strongly reactive (>10) S/CO by BPX or ARC assays by week 12 and week 24 on ART is summarized
137 in Table 1. After 12 weeks of therapy initiated in early infection, 69.6% of samples from individuals
138 who initiated ART at Fiebig I, 12.8% at Fiebig II, and 9.1% at Fiebig III/IV were non-reactive. Of
139 those samples that were reactive in the assays, 17.4% (FI) , 56.4% (FII) and 40.9% (FIII/FIV)
140 exhibited low S/CO (<10) responses in the BPX and ARC assays. By week 24, samples from 52.2%,
141 7.7% and 4.5% of individuals initiating therapy at FI, II and III/IV remained non-reactive, respectively.
142 Samples from only 10 of the 84 (11.9%) individuals initiating ART at FI-IV in our study, achieved the
143 very strong reactive response (>100 S/CO) as observed in untreated HIV-1 chronic infection by this
144 time.

145 The results of the BRC, ARC, and BPX Ag/Ab combo assays on samples from RV254/SEARCH010
146 participants who initiated ART at FI-IV and were tested after 24 weeks on therapy are shown in Figure
147 3. The three assays demonstrated overall good agreement at S/CO <10, while at higher reactivities, the
148 chemiluminescent ARC and fluorometric BPX assays yielded a broader dynamic range with S/CO
149 values to >200, compared to the colorimetric based BRC which saturates at a maximum S/CO of 12-
150 16. The ARC and BPX results were highly correlated to each other with a R2 of 0.8404. The BPX
151 assay, which in addition to HIV-1/2 reactivity also provides independent measures of the p24 Ag and
152 anti-HIV Ab detection for each sample, showed that reactivity, as expected, is due to the anti-HIV Ab
153 component alone, with no p24 Ag detected in any samples at week 12 or 24.

154 A more detailed picture of the relative performance of the BRC, ARC, and BPX assays at 24 weeks
155 after ART initiation as stratified by those who initiated therapy at FI, FII or FIII/IV is shown in Figure
156 4. By 2-4 weeks of infection, samples from untreated HIV infected individuals consistently show
157 S/CO values near the maximum range of the BRC (12-16) assays and 1/2/O (10-13), respectively, and
158 remained high thereafter (see Fig 1). In contrast, more than half (12 of 23) of the samples from

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159 individuals initiating ART at Fiebig I remained non-reactive (S/CO <1) on all three Ag/Ab combo
160 assays at week 24. An additional 8 samples demonstrated very low S/CO (<10) reactivity by ARC and
161 BPX assays, with those from only two individuals demonstrating S/CO of >100. Samples from all
162 individuals treated at Fiebig II and III/IV were reactive on all three assays prior to treatment initiation
163 (at Week 0), but by 24 week on ART, samples from 3 of the 39 FII individuals and one of the 22
164 FIII/IV individuals were non-reactive by all three assays.

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165 The ability of supplemental, confirmatory assays to confirm HIV reactivity in samples from
166 individuals who initiated ART during AHI was evaluated using the Geenius and HIV-1 WB assays.
167 HIV-1 WB profiles of all 15 of 30 of untreated individuals tested demonstrated typical HIV-1 WB
168 seroconversion profiles. An example for an untreated individual is shown in Figure 5: Reactivity to
169 each antigen increases with time yielding a mature, florid HIV-1 WB profile at weeks 11 and 21. In
170 contrast to untreated HIV-1 infected individuals who seroconverted to HIV-1 WB positivity by 7
171 weeks post first Aptima RNA reactivity, individuals who initiated ART at FI-IV showed a much
172 slower evolution of HIV-1 WB antigen reactivity. The HIV-1 WB demonstrated only faint antigen
173 reactivity by 24 weeks on ART which in many cases was insufficient for interpretation as a positive
174 result. As shown by the examples in Figure 5, a number of individuals initiating ART during early
175 stages of AHI were either negative or indeterminate on HIV-1 WB after 12 and 24 weeks on ART.
176 Although specific HIV-1 WB patterns were variable among individuals, even those who scored HIV-1
177 WB Positive, failed to develop the expected florid antigen reactivity typically observed in untreated
178 individuals.

179 The reactivity to HIV p24, p31, gp41, and gp160 antigen as detected by Geenius and WB at 24 weeks
180 after ART initiation in early infection is shown in Figure 6. The reactivity of all antigens is
181 significantly inhibited when ART is initiated in Fiebig I. The reactivity to gp41 in samples from
182 individuals initiating therapy in FI-IV is comparable in Geenius and HIV-1 WB assays, while detection
183 of gp160 (p=0.005), and p24 (p<0.0001) at each Fiebig Stage is significantly lower by Geenius than
184 HIV-1 WB. Reactivity to p31, a late appearing antigen, is either low or negative in both Geenius and
185 HIV-1 WB assays at 24 weeks after early ART initiation.

186 The decreased antigen reactivity translates to a high percentage of early treated individuals remaining
187 negative or indeterminate by Geenius and HIV-1 WB supplemental confirmatory tests after 12 and 24

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188 weeks of treatment (Table 2). At 12 weeks of ART, the Geenius assay was positive in only 25% of
189 samples from FI individuals, 44.7% of FII, and 59.1% of FIII/IV, while remaining samples were
190 negative or indeterminate. Similar results were observed at week 24, with samples from 26.1%, 50.0%
191 and 45.5% of FI, FII, and FIII/IV individuals, respectively, positive by Geenius. HIV-1 WB detection
192 sensitivity was somewhat higher with 30.4% (FI), 73.7% (FII) and 59.1% (FIII/IV) samples yielding a
193 positive result at week 24.

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194

195 Discussion
196 Previous results of the RV254/SEARCH010 study showed a marked delay in appearance or absence of
197 serological markers of HIV-1 infection in serial samples from individuals who initiated ART during
198 AHI (18). The present study extends the evaluation of samples from this cohort to examine the impact
199 of early ART on reactivity of newer HIV Ag/Ab combo, and supplemental, confirmatory assays used
200 to generate laboratory evidence of HIV infection. The detection of serological markers for HIV-1
201 infection in samples from ART treated individuals was contrasted to those from untreated individuals
202 from the RV217 Early HIV Cohort study (21, 22).

203 As demonstrated in the RV217 cohort of early HIV infection, the first antigen marker appears very
204 early in infection with the emergence of p24 Ag which is detected by Ag/Ab combo assays. Antigen
205 concentration as measured by Bio-Rad p24 Ag assay reaches maximum concentration at approximately
206 two weeks after first RNA detection, then rapidly declines following emergence of anti-HIV-1 Ab.
207 The p24 Ag in all samples from RV254 individuals at FII-IV was detectable prior to ART but was non-
208 detectable within one week on ART and remained non-detectable thereafter. Since p24 Ag is no
209 longer detectable in untreated individuals after 4-5 weeks of infection, or after one week of therapy, the
210 high S/CO values observed by the Combo assays is due to the Ab component only, as exhibited by
211 1/2/O or the Ab signal in the BPX assay.

212 Plasma from untreated individuals generated a S/CO at the maximum range for both BRC and 1/2/O
213 assays within 3-4 weeks of first Aptima RNA reactivity. HIV-1 WB antigen profiles continued to
214 increase in intensity with time, yielding a mature, florid HIV-1 WB pattern by Week 24. Although
215 testing by BPX or ARC was not performed on these samples at week 24, results from other studies

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216 have shown that the intensity of BPX and ARC results continues to increase over this time frame (32-
217 34).

218 Previous studies examining the ability of HIV Ag/Ab combo assays to detect AHI showed comparable
219 performance when BRC, BPX and ARC assays where used. The limit of detection of p24 Ag by these
220 assays ranged from 2-7 pg, which corresponds to 4.4 to 4.9 log copies RNA per ml and demonstrated a
221 reduction in the serological window period from 7 to 11 days earlier than the anti-HIV IgM/IgG

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222 immunoassays (28-31). In our study, the test results of BRC, ARC and BPX assays from samples
223 collected at 24 weeks after initiation of ART in AHI also showed generally good agreement at S/CO
224 results below 10. Whereas the BRC assay optical density read out saturates quickly at S/CO of 12-16,
225 the ARC and BPX assays provided a useful measure of the impact of early treatment on evolution of
226 Ab reactivity. We show that the high S/CO values typically seen in ARC (>700) and BPX (>200)
227 assays by 12-24 weeks in untreated individuals (32-34) were greatly suppressed by early ART.
228 Although the most dramatic inhibition of serologic reactivity at weeks 12 and 24 post initiation of ART
229 was observed when treatment was initiated in FI, a significant number of samples from individuals
230 treated at FII-IV also demonstrated lower reactivity by ARC and BPX assays.

231 A decrease in Ab detection after early treatment was also observed in other studies, which showed
232 increased frequencies of false-negatives, weakly-reactive HIV rapid tests, and increased numbers of
233 indeterminate HIV-1 WB results (11, 35). Of note, S/CO of less than 10 by ARC and BPX assays
234 have been frequently associated with false positive results (34, 36-38). The lower Ab detection in
235 ART treated individuals also led to increased estimates of recent infection when tested on Ab avidity-
236 based HIV incidence assays, such as the Limiting Antigen (LAg), Bio-Rad Avidity, and HIV-1
237 Multiplex assays, which rely on semi-quantitative detection of the intensity of the immune response
238 (32, 39).

239 Previous studies on the performance of the Geenius assay showed comparable performance relative to
240 the HIV-1 WB for detection of AHI (40, 41). In contrast, our results show that the HIV-1 WB is more
241 sensitive than Geenius for confirmation of infection in individuals treated in acute infection.
242 Examination of reactivity to individual antigens detected by Geenius assay in ART treated individuals,
243 showed that this assay is less sensitive than HIV-1 WB for detection of p24 and gp160 (Figure 6); two
244 antigens important for a positive result interpretation with this assay. Our results show that antigen

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245 reactivity by both Geenius and WB fails to develop after early ART treatment. A high percentage of
246 individuals remain negative or non-reactive by these confirmatory assays at week 12 and 24. The
247 decrease in HIV-1 WB reactivity observed at 24 weeks after start of treatment in FII (73.7%) vs
248 FIII/IV (59.1%) (Table 2) may be due to the small sample size. Statistical analysis showed no
249 significant difference (p = 0.243) between the percent HIV-1WB positive at FII vs FIII/IV at week 12
250 or week 24. The differences in the percent Geenius positive were smaller and was also not significant.

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251 These results are similar to those of a previous study which found that the p24 and p31 antigens in
252 chronic HIV infection were less frequently detected by Geenius than by HIV-1 WB or INNO LIA, and
253 that Geenius was less suitable for distinguishing between acute and chronic infections (42). Our
254 finding that 73.9% of individuals who initiated therapy at FI, and over 50% of those at FII, FIII/IV
255 would not have been confirmed by Geenius assays present particular diagnostic challenges, especially
256 in the absence of HIV-1 RNA in individuals under ART.

257 The impact of delayed evolution of serological response as manifested by weaker reactivity on
258 screening and confirmatory assays will have important implications in evaluating the infection status
259 of individuals who initiate ART in AHI or following suspected exposure to HIV. The high rates of
260 false negative Rapid Device Tests of HIV-1 infected children on long-term ART can lead to
261 misclassification, inadequate patient management, and/or the false perception of lack of infection or
262 cure (43). Frequent false negative serological results were reported in HIV-1-infected infants who
263 were breast-fed by mothers on ART or in post-exposure prophylaxis (PEP) programs to prevent
264 mother-to-child transmission (44-46). A significant reduction in HIV Ab levels, including lack of
265 HIV-1 WB reactivity and increase of false negative serological results following ART initiation had
266 previously been reported in other studies (9, 47, 48). Individuals from the RV254 cohort described in
267 this study who initiated treatment during FI remained HIV-1 infected despite full suppression of HIV-1
268 plasma viremia with no detectable serological markers for over a 2-year period, as HIV RNA rebound
269 occurred when ART was discontinued (49).

270 Despite the high success and continued expansion of ART-based prevention and treatment programs
271 such as Treatment as Prevention, Pre-Exposure Prophylaxis (PrEP), and Post Exposure Prophylaxis
272 (PEP), HIV breakthrough infections can still occur due to non-adherence or emergence of drug
273 resistant strains. Serological and nucleic acid assays used in current HIV diagnostic algorithms may

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274 not be sufficient to accurately identify true infection in settings where antiviral treatment is provided
275 very early in HIV infection. Individuals who become infected while on PrEP have slower Ab avidity
276 maturation, prolonged seroconversion profiles and an increase in time (from 49 to 80 days) to full
277 HIV-1 WB positivity (50, 51). Only 27/57 (47%) of RNA positive individuals who were infected
278 while on PrEP, were found reactive by Bio-Rad GS HIV Combo Ag/Ab EIA and none were HIV-1
279 positive by either HIV-1 WB or Geenius (52). Alternative approaches such as testing for cell-

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280 associated HIV total nucleic acid (RNA and DNA) may be required to rule out HIV-1 infections in
281 individuals with inconclusive HIV diagnostic test results and may be helpful to confirm infections in
282 such cases (6, 53).

283 Disclaimer
284 The views, opinions and/or findings contained in this report are those of the authors and should not be
285 construed as an official Department of the Army position, policy or decision unless so designated by
286 other documentation. The content of this manuscript is solely the responsibility of the authors and
287 does not necessarily represent the official views of any of the institutions mentioned above, the U.S.
288 Department of the Army or the U.S. Department of Defense, the National Institutes of Health, the
289 Department of Health and Human Services, or the United States government and the Thai Red Cross
290 AIDS Research Centre.

291 Conflict of interest

292 JA has received honoraria for participating in advisory meetings for ViiV Healthcare, Gilead, Merck,
293 Roche and AbbVie. Other authors have no conflict of interest.

294 Acknowledgements and funding

295 We are grateful to the participants and study volunteers for their contribution and commitment to make
296 this HIV research possible. This work is supported in part by the US Army Medical Research and
297 Materiel Command under Contract No.W81-XWH-18-C-0337 and W81-XWH-16-C-0225 and by the
298 US Military HIV Research Program, the Walter Reed Army Institute of Research under a cooperative
299 agreement (W81-XWH-07-2-0067, W81-XWH-11-2-0174) with the Henry M. Jackson Foundation for
300 the Advancement of Military Medicine, Inc. and by the NIH grant R01AI108433 between the Henry
301 M. Jackson Foundation for the Advancement of Military Medicine, Inc., and the U.S. Department of
302 Defense (DoD), and by the Division of AIDS, the U.S. National Institute of Allergy and Infectious

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303 Diseases, and by an intramural grant from the Thai Red Cross AIDS Research Center. The US Army
304 Medical Research Acquisition Activity (820 Chandler Street, Fort Detrick, MD 21702-5014, USA) is
305 the awarding and administering acquisition office for the cooperative agreement. Antiretroviral therapy
306 was supported by the Thai Government Pharmaceutical Organization, Gilead, Merck and ViiV
307 Healthcare.

308 The RV254/SEARCH010 Study Team: from SEARCH/TRCARC: Praphan Phanuphak, Nipat

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309 Teeratakulpisarn, Eugene Kroon, Carlo Sacdalan, Phillip Chan, Ponpen Tantivayakul, Nitiya
310 Chomchey, Ratchapong Kanaprach, Jintana Intasan, Duanghathai Sutthichom, Peeriya Prueksakaew,
311 Pacharin Eamyoung, Suwanna Puttamaswin, Khunthalee Benjapornpong, Somporn Tipsuk, Tassanee
312 Luekasemsuk, Kultida Poltavee; from Chulalongkorn University: Supranee Buranapraditkun, Sunee
313 Sirivichayakul, Phandee Wattanaboon-yongcharoen; from AFRIMS: Robert O’ Connell, Alexandra
314 Schuetz, Siriwat Akapirat, Rapee Trichavaroj, Bessara Nantapinit, Numpueng Churikanont, Saowanit
315 Getchalarat, Nongluck Sangnoi; from MHRP: Merlin Robb, Trevor Crowell, Suteeraporn Pinyakorn,
316 Ellen Turk, Oratai Butterworth, Corinne McCullough, Sodsai Tovanabutra, Lydie Trautmann, Mark
317 Milazzo.

318 RV 217 Study Teams: Hannah Kibuuka (Uganda), Lucas Maganga (Tanzania), Sorachai Nitayaphan
319 (Thailand), Fred K Sawe (Kenya).

320 The views expressed are those of the authors and should not be construed to represent the positions of
321 the U.S. Army or the Department of Defense.

322

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515

516 Figure and Table Legends

517

518 Figure 1. Time course of evolution of serological markers in untreated HIV-1 infected individuals
519 (RV217). A) Reactivity of 30 untreated HIV-1 infected individuals by the Bio-Rad GS Ag/Ab Combo
520 (BRC) which measures both p24 antigen and anti-HIV antibody. B) Average (S/CO values for BRC
521 and 1/2/O; pg/ml of p24 Ag as measured by the Bio-Rad p24 Ag Assay; and percent of individuals
522 with HIV-1 Western Blot Positive (WB) samples at each time point.

523

524 Figure 2. Evolution of BioPlex (BPX) or Architect (ARC) reactivity of individuals initiating ART
525 during Acute HIV infection. Solid lines represent BPX results, with very similar trends seen with
526 ARC (Dotted lines) The ARC are shown only results for those individuals for whom insufficient
527 sample was available for BPX testing at Week 0. Individuals depicted in green were non-reactive on
528 ARC or BPX by Week 24, while those in blue demonstrated low reactivity (S/CO <10), and those in
529 red strong reactivity(S/CO>10). Very few samples demonstrated the very strong reactive response
530 (S/CO >100) as seen in established infections from untreated individuals by 24 weeks of infection.

531

532 Figure 3. Comparison of reactivity of Architect (ARC), Bio-Rad (BRC) and BioPlex (BPX) Ag/Ab
533 Combo Assays in individuals who initiated ART at FI-IV and were tested after 24 weeks on ART.

534

Decreased Seroreactivity after ART 6/14/19 Page 18

535 Figure 4. Distribution of S/CO values at week 24 after initiation of ART at Fiebig I, II or III/IV by the
536 Bio-Rad (BRC), Architect (ARC), or BioPlex (BPX) HIV Ag/ab Combo assays. The horizontal bars
537 show geometric mean and standard deviation for each set.

538

539 Figure 5. Evolution of HIV-1 Western Blot (WB) profiles of an untreated HIV-1 infected individual

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540 with time after first Aptima reactive RNA sample (Left). HIV-1 WB profiles from selected individuals
541 at week 12 and 24 following initiation of ART at Fiebig I-IV stage of HIV infection (Right).

542

543 Figure 6. Reactivity of HIV antigens on Geenius (GN) and HIV-1 Western blot (WB) assays at 24
544 weeks after initiation of therapy at FI (green), F2 (yellow) and FIII/IV (blue).

545

546 Table 1. Percentage of samples from individuals with non-reactive (S/CO<1.0), weakly reactive
547 (S/CO<10) or strongly reactive (S/CO>10) results on the BioPlex or Architect assays at week 12 and
548 24 after initiation of therapy at Fiebig I to IV.

549

550 Table 2. Positivity of the HIV-1 Western Blot (WB) and Geenius (GN) assays at 12 and 24 weeks
551 after initiation of ART at Fiebig I, II or III/IV. A high percentage of samples tested at 12 and 24 weeks
552 of ART remain negative or indeterminate when therapy is initiated early.

553

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 Downloaded from http://jcm.asm.org/ on August 6, 2019 by guest
A B 1000
100
3

100
120

p 24 A g

W B

p 2 4 A g (p g /m l)
100
B R C 100%
B R C
A v e . s /c o

10 10
80
1 /2 /O 100 80%
s /c o

%
2

W B P o s itiv e
60
60%

c u t-o ff 1 14 0 40%

1 10
20
20%

0 .1 0 .1 0
00 77 1144 2211 22 88 33 55 44 22 44 99
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7

W e e k s a fte r fir s t A P T IM A
 Downloaded from http://jcm.asm.org/ on August 6, 2019 by guest
F ie b ig I F ie b ig II F ie b ig III/IV
1000 1000 1000

100 100 100

s /c o

>10

10 10 10

<10

c u t-o ff 1 1 1

< 1 .0

0 .1 0 .1 0 .1

0 12 24 0 12 24 0 12 24

W eeks o f A R T
 Downloaded from http://jcm.asm.org/ on August 6, 2019 by guest
A R C vs B R C B P X vs B R C B P X vs A R C
1000
1000 1000
c u t-o ff

2
R = 0 .8 4 0 4

100
100 100
A R C s /c o
B R C s /c o

B R C s /c o

10 10
10

c u t-o ff 1
1 1

0 .1
0 .1 0 .1
0 .1 1 10 100 1000
0 .1 1 10 100 1000 0 .1 1 10 100 1000

A R C s /c o B P X s /c o
B P X s /c o
 Downloaded from http://jcm.asm.org/ on August 6, 2019 by guest
1000 1000 1000
1 ,0 0 0

FI F II F III/IV N o A R T
100 100 100
100
s /c o

10 10 10 10

c u t-o ff 1 1 1 1

0 .1 0 .1 0 .1 0 .1

B R C A R C B P X B R C A R C B P X B R C A R C B P X B R C A R C B P X
Downloaded from http://jcm.asm.org/ on August 6, 2019 by guest
Downloaded from http://jcm.asm.org/ on August 6, 2019 by guest
WB
gp160

GN
WB
gp41

GN
WB
p31

GN
WB
p24

GN
100%

80%

60%

40%

20%

0%
% Reactive Antigens
Table 1

Week 12 Week 24
FI FII FIII/IV FI FII FIII/IV
NR 69.6 12.8 9.1 52.2 7.7 4.5
S/CO <10 17.4 56.4 40.9 34.8 38.5 36.4

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S/CO >10 13.0 30.8 50.0 13.0 53.8 59.1
Total 100 100 100 100 100 100
Table 2
Week 12 Week 24
FI FII FIII/IV FI FII FIII/IV
WB GN WB GN WB GN WB GN WB GN WB GN
POS 25.0 25.0 65.8 44.7 72.7 59.1 30.4 26.1 73.7 50.0 59.1 45.5
IND 37.5 20.8 31.6 52.6 27.3 40.9 21.7 21.7 21.1 44.7 36.4 45.5
NEG 37.5 54.2 2.6 2.6 0.0 0.0 47.8 52.2 5.3 5.3 4.5 9.1

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100 100 100 100 100 100 100 100 100 100 100 100