JCM Accepts, published online ahead of print on 2 September...

Soft Endpoint for HIV Growth in Human Tissue Explants For Journal of Clinical Microbiology

Page 1 of 44

Multi-site comparison of anti-HIV microbicide activity in explant assays using a novel 1

endpoint analysis 2

3

Nicola Richardson-Harman1ı

, Carol Lackman-Smith1*, Patricia S. Fletcher

2, Peter A. 4

Anton3, James W. Bremer

4, Charlene S. Dezzutti

5, Julie Elliott

3, Jean-Charles Grivel

6, Patricia 5

Guenthner7

, Phalguni Gupta8, Maureen Jones

1, Nell S. Lurain

4, Leonid B. Margolis

6, Swarna 6

Mohan5, Deena Ratner

8, Patricia Reichelderfer

6, Paula Roberts

1, Robin J. Shattock

2, and James 7

E. Cummins Jr.1 8

9

Microbicide Quality Assurance Program, Southern Research Institute, Frederick, MD1, Centre 10

for Infection, Department of Cellular and Molecular Medicine, St. Georges University of 11

London, London, UK2, Center for Prevention Research, University of California, Los Angeles, 12

CA3, Virology Quality Assurance Laboratory, Department of Immunology and Microbiology, 13

Rush University Medical Center, Rush University, Chicago, IL4, Magee-Women’s Research 14

Institute, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of 15

Pittsburgh, Pittsburgh, PA5, Program in Physical Biology, Eunice Kennedy-Shriver National 16

Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD6, 17

National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease 18

Control, Atlanta, GA7, and Department of Pathology, School of Medicine, University of 19

Pittsburgh, Pittsburgh, PA8

. 20

21

ıCurrent affiliation: Alpha StatConsult LLC, Damascus, MD. [email protected]. 22

Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00673-09 JCM Accepts, published online ahead of print on 2 September 2009

on May 22, 2018 by guest

http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 2 of 44

*Corresponding author: Carol Lackman-Smith, Southern Research Institute, 431 Aviation Way, 1

Frederick, MD 21701; 301-694-3232 (Tel.); 301-694-7223 (Fax); 2

[email protected] 3


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 3 of 44

ABSTRACT 1

Microbicide candidates with promising in vitro activity are often advanced for 2

evaluations using human primary tissue explants relevant to the in vivo mucosal transmission of 3

HIV-1, such as tonsil, cervical or rectal tissue. To compare virus growth or the anti-HIV-1 4

efficacy of candidate microbicides in tissue explants, a novel ‘soft endpoint’ method, was 5

evaluated to provide a single, objective measurement of virus growth. The applicability of the 6

soft endpoint is shown across several different ex vivo tissue types, performed in different 7

laboratories, and for a candidate microbicide (PRO 2000). The soft endpoint was compared to 8

several other endpoint methods including: 1) the growth of virus on specific days after infection; 9

2) the area under the virus growth curve; and 3) the slope of the virus growth curve. Virus 10

growth at the assay soft endpoint was compared between laboratories, methods and experimental 11

conditions using non-parametric statistical analyses. Intra-assay variability determinations using 12

the coefficient of variation demonstrated higher variability for virus growth in rectal explants. 13

Significant virus inhibition by PRO 2000 and significant differences in the growth of certain 14

primary HIV-1 isolates were observed by the majority of laboratories. These studies indicate that 15

different laboratories can provide consistent measurements of anti-HIV-1 microbicide efficacy 16

when: (i) the soft endpoint or other standardized endpoint is used; (ii) drugs and/or virus reagents 17

are centrally sourced and; (iii) the same explant tissue type and method are used. Application of 18

the soft endpoint reduces the inherent variability in comparisons of pre-clinical assays used for 19

microbicide development. 20

21

22

23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 4 of 44

1

Introduction 2

3

Studies using human tissues cultured ex vivo (i.e. tissue explants) are performed for the 4

pre-clinical evaluation of topical microbicides, compounds that can be applied vaginally or 5

rectally to reduce the sexual transmission of HIV-1 (21). Various tissue explant models and the 6

procedures used for evaluating test compounds for anti-HIV activity have been described (1, 2, 7

4, 9-11, 15, 16). At present there is no standard methodology, including calculation of an 8

endpoint, for comparing results between ex vivo experiments. 9

Although cell-based assays can be standardized (6, 22), tissue explants may represent a 10

more relevant method of testing since this is where HIV-1 infection occurs in vivo. As most 11

parameters of cell-based assays (i.e. size of virus inoculum, number of target cells, and use of a 12

single endpoint measure) can be standardized, the data are often less variable and more easily 13

reproduced in comparison to assays with tissue explants. For example, the number of target cells 14

in cell based assays can be controlled, whereas the concentration and distribution of target cells 15

in explant tissues are highly variable. In addition, explant methods vary across laboratories and, 16

as a consequence, a number of parameters may affect the reliability of the results: (i) tissue type; 17

(ii) HIV-1 strain or isolate ; (iii) culture medium formulation; (iv) size of virus inoculum; (v) 18

length of virus incubation; (vi) frequency of medium change; (vii) concentration of test 19

compound; (viii) drug treatment period prior to or after viral exposure; (ix) use of controls; and 20

(x) endpoint viral growth measurements. 21

For the reasons described above, a group of microbicide investigators joined a mutual 22

effort to identify an approach for improving the reliability and reproducibility of data from 23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 5 of 44

explant studies. To this end, results obtained from different explant models were analyzed using 1

a range of endpoint methods. In general, due to the inter-dependency of repeated measurements, 2

standard statistical methods (e.g. t-test, ANOVA, Mann Whitney and Kruskal-Wallis) do not 3

apply when comparing virus growth in the presence of inhibitory compounds. For example, high 4

virus growth on one day in one explant sample is likely to continue to be followed by high virus 5

growth throughout the assay period, while low virus growth in a different sample will be 6

followed by low growth. In some cases the relationships between consecutive data points follow 7

certain assumptions, thus allowing statistical modeling techniques to be applied (e.g. repeated 8

measures ANOVA, generalized linear and mixed models (28)). However, this type of statistical 9

modeling may be outside the expertise of many microbiological research teams, and the inherent 10

assumptions of normal distributions and homogeneity of variance are rarely met for virus growth 11

data. The soft endpoint (SOFT) is presented as a single, summary measure of virus growth to 12

enable direct comparisons between explant experiments. 13

There is currently no consensus on the method to determine a summary measure of virus 14

growth (27). A number of methods including the area under the virus growth curve [AUC, (24)], 15

the slope of the virus growth curve (36) and virus growth at specific time points (1, 9, 10, 17) 16

have all been used. The first aim of this study was to compare a selection of alternative, single 17

measures of virus growth, including SOFT, using data from multi-site explant studies as part of a 18

wider effort to improve the quality of explant studies by standardizing the method used to 19

objectively measure HIV-1 replication in tissues (5, 32). 20

The second aim of this study was to apply SOFT to compare treatment conditions 21

relevant to microbicide research using the explant methods developed by the participating 22

microbiological laboratories. It should be noted that the goal of this work was to identify those 23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 6 of 44

key parameters that would improve overall reliability and reproducibility of explant studies as 1

opposed to having the investigators standardize every aspect of their protocols. Seven 2

laboratories used their preferred human tissue type and explant method to infect with an in-house 3

or commonly sourced HIV-1 stock in the presence or absence of the same commonly sourced 4

drug. The null hypothesis of no effect of the various treatment conditions on virus growth was 5

tested. When the null hypothesis was rejected, similarities in statistically significant treatment 6

effects across explant methods provided evidence of inter-laboratory and/or inter-method 7

reproducibility in explant experimental results. Conversely, differences in treatment effects 8

across explant methods indicated a need for either standardization of explant methodology or 9

thorough characterization of the observed differences. A multi-site study design was used to 10

evaluate the conditions that could affect virus growth in explants, and subsequent interpretation 11

of the efficacy of a candidate microbicide (PRO 2000). These variables included: (i) source/type 12

of assay reagents (virus stocks and medium); (ii) tissue type (cervical, rectal and tonsil); (iii) how 13

the tissue was cultured; (iv) HIV isolate (HIV-1Ba-L and clinical isolates of differing clades); and 14

(v) PRO 2000 concentration. 15

16


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 7 of 44

Materials and Methods 1

2

Virus. The central virus stocks were provided by the Division of AIDS Viral Quality 3

Assurance Laboratory (VQA LAB). Each laboratory was sent central stocks of HIV-1Ba-L (VQA 4

Ba-L or VQAB) to be compared to the usual laboratory HIV-1Ba-L stock (in-house Ba-L or IHB). 5

Laboratories were also sent aliquots of four clinical HIV-1 isolate subtypes: UG273 (Clade A); 6

DJ263 (Clade A/G recombinant); SE364 (Clade C); and UG268 (Clade C). All viral stocks were 7

kept at -70°C and thawed to room temperature. Immediately after thawing, the contents of each 8

vial were mixed and the vial was placed on wet ice prior to use. Cervical, rectal and tonsil tissue 9

explants were exposed, overnight, to VQAB or viral subtypes at 104, 10

3 or 10

3 TCID50, 10

respectively. IHB stocks were tested at a 104 TCID50 for all laboratories with the exception of 11

laboratory C, where tissue was exposed to 5×104 TCID50. The laboratories were asked to use 12

virus titers in their explant tissues that routinely gave sufficient levels of infection in their virus 13

controls. Thus tissue explants were inoculated with identical, centrally distributed samples of 14

HIV-1Ba-L at a TCID50 of 103 for tonsil and rectal tissue and 10

4 for cervical tissue, based on 15

previously established HIV-1 susceptibility of these tissues. 16

Media. All laboratories were asked to test a common medium (VQA LAB medium or 17

VQAM) alongside their usual in-house medium (IHM). The different media used are described 18

in Table 1. (1, 2, 4, 10, 11, 13, 17-19). 19

Microbicide Candidate. Each laboratory was supplied with an aqueous stock of PRO 20

2000 (40mg/ml), an unformulated naphthalene sulfonate polymer, generously provided by Endo 21

Pharmaceuticals Solutions, Inc. (Lexington, MA). PRO 2000 was diluted using culture media 22

and tested at 5, 50 and 500たg/ml (final concentrations). 23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 8 of 44

Explant tissue. All human tissue samples were collected under Institutional Review 1

Board approved protocols at each participating institution. Between one and three donor samples 2

of cervical (Laboratories A, B, C, E and G), tonsil (Laboratory D), and rectal (Laboratory F) 3

tissues were used by each laboratory. Explant tissue punch sizes and polarization treatment are 4

listed in Table 1. 5

Explant assays. Each of the seven laboratories performed tissue infection and harvesting 6

of culture supernatants using their chosen tissue explant methods (Table 1). Each condition was 7

tested in duplicate-quadruplicate according to each laboratory’s protocol. For all laboratories, 8

supernatants were sampled following viral exposure before washing, at the final wash following 9

virus removal, and then every 2-3 days during culture for a maximum of 15 days. Supernatants 10

were stored at -20flC, with the exception of Laboratory C where supernatants were stored at -11

80°C, prior to evaluation for viral infection by the presence of p24 by ELISA. 12

Study design. Using their usual tissue explant method, all participating laboratories 13

completed three sub-studies as follows: (i) Source of virus/media, evaluated IHB/IHM, 14

VQAB/IHM, IHB/VQAM, VQAB/VQAM; (ii) Evaluation of a candidate microbicide where 15

VQAB was compared to VQAB + PRO 2000 (5, 50 or 500og/ml); and (iii) Susceptibility of 16

tissue explants to different HIV-1 isolates where VQAB was compared to UG273 (Clade A), 17

DJ263 (Clade A/G recombinant), SE364 (Clade C) and UG268 (Clade C). Each laboratory was 18

asked to submit raw optical density readouts (ODs) and calculated p24 results for statistical 19

analysis. In addition, for the (i) Source of virus/media study the participating laboratories were 20

requested to dispatch matched aliquots of culture supernatants for independent determination of 21

p24 levels by the VQA LAB laboratory. 22

23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 9 of 44

Statistical Methods 1

Definition of the Soft Endpoint. The soft endpoint (SOFT) is presented as a cross-2

sectional index from a growth curve that is reflective of the virus growth achieved in the assay. 3

SOFT was formulated to define the time-point at which rapid, and in some cases exponential, 4

rates of virus growth have been achieved and no further biologically significant increases in virus 5

growth are apparent. SOFT is the last time point when the increase in virus concentration 6

between two consecutive time points is greater than the square root of the sum of sequential 7

changes in virus concentration for the entire assay. 8

Growth of HIV-1 can be divided into four main phases: lag time (no growth), an 9

exponential growth phase, a stationary phase and a decline phase (12, 35). SOFT is presented 10

here as an estimation of the start of the stationary phase of virus growth, after the exponential 11

growth phase has been achieved and prior to the onset of the decline phase (12, 35). SOFT is 12

defined as the last time point (k) where; 13

14

SOFT can be described as ‘one-way’, as it is set only by significant increases in virus 15

growth (see Supplement 1). 16

Comparison of SOFT to other endpoint methods. A variety of endpoints were 17

determined for growth curves from a sub-sample of assays using centrally provided virus and in-18

house media (‘VQAB/IHM’, see Supplement 2). The VQAB/IHM assays were run over 12-15 19

days where endpoints compared virus growth (p24 pg/mL) at three time points : (i) SOFT, (ii) 20

day 12 (DAY12), (iii) day 15 (DAY15); and two parameters of the growth curve: (iv) The slope 21

of the virus growth curve (SLOPE) calculated using a second order polynomial equation (PROC 22

Äp24 (Timek-Timek-1) > Äp24 ¬ABS(Timek-Timek-1)


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 10 of 44

NLIN; SAS/STAT®

, Version 9.1); and (v) the area under each virus growth curve (AUC) 1

approximated using the trapezoidal rule (37). It has been suggested to limit slope measurements 2

to the linear part of the virus growth curve, where the linear portion is determined by visual 3

inspection of each curve (36). As the aim of this research was to provide an objective single 4

measure of virus growth, a non-linear formula was applied to the entire growth curve to calculate 5

the slope value. The trapezoidal rule was used to estimate the AUC of the function f(x), by 6

calculating the total area of adjacent trapezoid shapes. SOFT, DAY12 and DAY15 endpoints 7

were the p24 concentrations measures made on these respective days, the SLOPE endpoint was a 8

measure of the rate of p24 production, and the AUC endpoint was a measure of total p24 9

production. Linear discriminant analysis [LDA, PROC STEPDISC; SAS/STAT®

, Version 9.1 10

(20, 30)], a multivariate statistical procedure, was used to test the ability of each endpoint to 11

predict low (p24 < 1,500 pg/ml), medium (1,500-20,000) and high (>20,000) virus growth from 12

the VQAB/IHM assays collected for study arm ‘Source of virus/media.’ LDA mathematically 13

defines a discriminant function to separate data into distinct groups. In this case, the data were 14

the assay endpoints and the groups were low, medium or high depending on the virus growth for 15

each assay. A stepwise LDA was performed to compare the ability of each of the assay endpoints 16

to predict virus growth. The median p24 levels for the endpoint measures of SOFT, DAY12, 17

DAY15 and AUC were compared for the low, medium and high virus growth assays using 18

Kruskal-Wallis analysis of variance with Dunn’s multiple comparison test (alpha=0.05). 19

SOFT used to calculate % virus inhibition. SOFT was applied to virus growth data 20

from drug treated and control conditions, where the drug was PRO 2000, a candidate anti-HIV-1 21

microbicide. Data are presented from a cervical explant experiment from one participating 22

laboratory where three cervical explant donor tissues were infected with HIV-1Ba-L in the 23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 11 of 44

presence of three concentrations of PRO 2000 (5, 50 and 500 たg/ml) and in the absence of PRO 1

2000 (virus control). The SOFT for each donor was set using the averaged virus control results. 2

The % virus inhibition at each level of PRO 2000 was calculated using the formula below: 3

4

% virus inhibition = 100 * (treatment p24/ control p24) 5

6

The % virus inhibition was compared to 0% (i.e. control p24 at the soft endpoint) by 7

Wilcoxon Signed Rank Test. The variability in the % virus inhibition results using the SOFT, 8

DAY10 and DAY14 endpoints were compared by % coefficient of variation [%CV; 9

100*(standard deviation/mean)] for replicate measurements. The %CV provided an index of 10

variability, where a higher %CV indicates greater variability in measurements. 11

SOFT used to compare drug inhibition. SOFT was applied to calculate the inhibitory 12

effects of a potential microbicide, PRO 2000. The % virus control for PRO 2000 [(test sample 13

p24/Ba-L Control p24)*100] were calculated at the HIV-1Ba-L control soft endpoint. All % virus 14

control values œ 100% were converted to 100% virus control for the purpose of this study. Non-15

linear regression analysis (sigmoidal dose response curve) of the % virus control measurements 16

onto the log10 PRO 2000 concentration was used to calculate the half maximal inhibitory 17

concentration (i.e. IC50) of PRO 2000 for each laboratory. The effects of HIV viral isolate were 18

tested by comparing the log differences in p24 released from each isolate (UG268, SE364, 19

DJ263 and UG273) to HIV-1Ba-L. 20

The p24 pg/mL or Log10 pg/mL results were not normally distributed (Shapiro-Wilk 21

W=0.26-0.95; P<0.0001), a pre-requisite for statistical tests such as ANOVA and t-test . The 22

non-parametric counterparts of the t-test (i.e. Mann Whitney, Wilcoxon Signed Rank Test) and 23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 12 of 44

ANOVA (Kruskal Wallis with Dunn’s multiple comparison test) were used to test the effects of 1

reagent source, explant method, microbicide concentration and HIV viral isolate on p24 release 2

at the assay soft endpoint. Non-parametric statistics are routinely used to compare such non-3

normally distributed data as HIV RNA (23) and HIV-1 p24 (33). All comparisons of treatment 4

effects to HIV-1Ba-L control were made within donor and all statistical analyses were performed 5

using SASTM

(Version 9.1; alpha=0.05). 6

7

Results 8

9

Seven laboratories, each using the same HIV-1 stock in their respective tissue explant 10

assays, collected supernatants for up to 15 days following virus exposure and performed a p24 11

ELISA to quantify virus replication. Participating laboratories submitted p24 results for each 12

arm of the study: (i) source of HIV-1Ba-L and media (laboratories A, C-G); (ii) concentration of 13

PRO 2000 (A-G); and (iii) isolate type (A-G). In-house calculated p24 values were equivalent to 14

those measurements made by a central laboratory (Pearson r = 0.82); and the effect of standard 15

curve formula used by the laboratories (e.g. linear vs. non-linear) was found to only effect low 16

p24 measurements (<100pg/mL; see Supplement 3) thus, laboratory calculated p24 values were 17

used in this analysis. SOFT was applied to all assays using laboratory calculated p24 values. 18

Comparison of Single Measures of Virus Growth. The VQAB/IHM assays from un-19

stimulated cervical tissue (Labs A, E and G), stimulated cervical (C), tonsil (D) and rectal (F) 20

were used to compare SOFT to a selection of growth curve endpoints from the ‘source of HIV-21

1Ba-L and media’ study arm (see Supplement 2). VQAB/IHM assays were grouped according to 22

the maximum p24 (MAXp24) achieved during the 15 days of culture into low (Figure 1a), 23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 13 of 44

medium (Figure 1b) and high (Figure 1c) virus growth. The soft endpoint, for each VQAB/IHM 1

virus control assay, is presented (Figure 1a-c). The virus growth profiles varied widely in the 2

different tissue explant models with SOFT ranging from day 3 to day 15 (Figures 1a-c). 3

The endpoint measures of SOFT, DAY12, DAY15 and AUC were compared for the 4

low, medium and high growth VQAB/IHM assays (Figure 2). The AUC resulted in larger p24 5

concentration ranges [median (IQ range) = 4.63×107(4.97×10

9) pg/ml] compared to the SOFT 6

[6.84×103(4.94×10

6) pg/ml], DAY12 [3.90×10

4(5.38×10

5) pg/ml] and DAY15 7

[2.28×103(3.38×10

5) pg/ml] endpoint measures (H(5) = 35.29, p<0.01, Figure 2) where a larger 8

IQ range reflects higher inter-assay variability . The high p24 result for the AUC endpoint was 9

expected as the AUC is an estimation of the total virus growth throughout the duration of the 10

experiment, in contrast to the other endpoint methods where the growth on specified days 11

(SOFT, DAY12 and DAY15) was calculated. There were no significant differences between 12

median p24 results for the DAY12, DAY15 and SOFT endpoints (P>0.05, Figure 2). The utility 13

of SLOPE, AUC, DAY12 and DAY15 as predictors of low, medium and high p24 yielding 14

assays were reduced for the medium yield assays, for which endpoints overlapped with both low 15

and high yielding assays, whilst the SOFT endpoint resulted in a clear separation between low, 16

medium and high yielding for the sub-sample of VQAB/IHM assays (Figure 2). 17

LDA was used to test the ability of each endpoint to predict low, medium and high 18

growth for the sub-sample of VQAB/IHM assays, where a high, significant R2 value describes 19

greater accuracy in prediction. The LDA procedure assumed a multivariate normal distribution 20

of endpoint measures within each assay yield group (20). Since it has been recommended to log 21

transform certain types of biological data prior to parametric analyses (34), p24 (pg/mL) at the 22

SOFT endpoint was entered into the LDA analysis as both a linear and a log transformed 23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 14 of 44

variable. The results of the LDA are presented in Table 2, where the significance level (P value) 1

of an F-test from an analysis of covariance (ANCOVA) indicated the ability of each endpoint to 2

predict assay virus growth. The endpoints that were not the focus of the analysis were entered 3

into the ANCOVA as covariates. The squared partial correlations for each endpoint are given 4

(R2, Table 2), controlling for the effects of the other endpoints in the model. The SOFT and log 5

transformed SOFT endpoint metrics were the most effective predictors of low, medium and high 6

p24 yielding assay group assignment, whilst DAY15 was the least effective predictor of p24 7

yield (Table 2). 8

SOFT used to compare the effect of tissue type and culture model on HIV-1Ba-L 9

infection. The p24 at the assay soft endpoint is presented in Figure 3 across tissue types and 10

methods. Virus production was found to vary widely according to tissue type and explant culture 11

method (Kruskal Wallis Analysis of Variance, P<0.0001, Figure 3). Using all measurements of 12

HIV-1Ba-L growth (in the absence of PRO 2000) irrespective of virus/media source, release of 13

p24, at the soft endpoint, was lowest for the laboratories using (un-stimulated) cervical tissue and 14

highest for laboratories using either cervical tissue with PBMC co-culture or tonsil tissue. There 15

were no statistical differences in virus yield between stimulated cervical explants when 16

compared to rectal or tonsil explants. Comparison of the three model systems used for cervical 17

tissue demonstrated that un-stimulated cervical tissue resulted in the lowest viral growth while 18

the use of a PBMC co-culture resulted in the highest p24 release. Stimulated cervical tissue 19

resulted in lower growth than the use of a PBMC co-culture but higher growth than un-20

stimulated cervical tissue. A similar trend was observed if only the best growth condition 21

(VQAB/IHM) was evaluated except that the difference between stimulated cervical tissue and 22

cervical tissue with PBMC co-culture was not statistically significant, and the difference in viral 23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 15 of 44

growth between rectal and cervical with PBMC co-culture/tonsil increased in statistical 1

significance (P<0.001; data not shown). 2

SOFT used to measure virus inhibition. An example of viral inhibition by PRO 2000 3

on HIV-1Ba-L growth is presented using un-stimulated cervical tissue explants (n=3) to 4

demonstrate the utility of SOFT in a compound screening program (Figure 4). Un-stimulated 5

cervical tissue explants, at SOFT, were found to have lower p24 production (median = 101.1 6

pg/ml) compared to rectal (626.9 pg/ml), tonsil (35,800 pg/ml), stimulated cervical (2,381 pg/ml) 7

and cervical with PBMC co-culture (139,950 pg/ml) explant models (P<0.0001, Kruskal Wallis 8

analysis of variance, Dunn’s Multiple Comparison test; Figure 3). As lower viral replication was 9

likely to result in a reduced ability to detect inhibition of virus, SOFT was used to calculate virus 10

inhibition in the un-stimulated cervical tissue. HIV-1Ba-L growth in the absence of drug, for two 11

donors (1 and 2), increased through days 3-10 but then decreased by day 14 (Figure 4a & b), 12

whilst in the third donor (donor 3), viral growth increased through days 7-14 (Figure 4c). SOFT 13

accommodated these inter-donor variations in virus growth by setting the time point for 14

comparison for donors 1 and 2 at day 10, prior to the decline in virus growth, and at day 14 for 15

donor 3, where delayed onset of virus replication was exhibited. Using SOFT, the % virus 16

inhibition ranged from 80.6-99.0% for the highest concentration of drug (500 µg/ml) across 17

replicate measurements and donors (Figure 4d), while a wider range in results were found using 18

either DAY10 (61.2-96.9%) or DAY14 (41.3-99.0%) endpoint measures. The % virus inhibition 19

results using all endpoints were more variable for the 5 and 50 たg/ml PRO-2000 conditions 20

which may be due to these concentrations being at the threshold of efficacy for this compound 21

(Figure 4d). Although a dose-response effect was found for % virus inhibition using all three 22

endpoint measures [Wilcoxon Signed Rank Test, P<0.05], less variability was observed using 23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 16 of 44

SOFT (%CV = 54.9) when compared to DAY10 (100.2%) and DAY14 (95.5%; Figure 4d). 1

Based on these results, SOFT was applied for the remaining comparisons. 2

SOFT used for evaluation of a candidate microbicide. The % virus inhibition of PRO 3

2000, a candidate microbicide, was calculated using the SOFT endpoint for donor-matched, 4

untreated HIV-1Ba-L control results for each laboratory and explant tissue type (Figure 5). With 5

the exception of stimulated cervical tissue explants, all tissue types demonstrated a dose-6

dependent effect of virus inhibition by PRO 2000 (Figure 5a). Significant viral inhibition was 7

observed at (i) 500og/ml PRO 2000 (cervical, cervical PBMC co-culture, rectal and tonsil; 8

P<0.001, Wilcoxon Rank Signed Test), (ii) 50og/ml PRO 2000 (un-stimulated cervical and 9

tonsil tissue, labs A, B and D, P<0.001; and un-stimulated cervical, lab E, P<0.01) and (iii) 5 10

og/ml PRO 2000 (un-stimulated cervical tissue, labs A and B, P<0.05; tonsil P<0.001). The 11

sigmoidal dose response curve explained between 24-81% of the variance across all explants 12

with the exception of the results from the cervical stimulated experiment where the data did not 13

conform to a dose response curve (Tables in Figures 5b & 5c). The IC50 of PRO 2000 varied 14

considerably across tonsil, cervical PBMC co-culture, rectal and stimulated cervical explants 15

(9.7-98.0 たg/ml; Figure 5b); greater consistency in IC50 measurements was found for the three 16

laboratories testing un-stimulated cervical explants (24.5-29.3 たg/ml Figure 5c). 17

Tissue responses vary according to HIV-1 isolates. The rate of viral growth of four 18

primary isolates of HIV-1 (i.e. UG268, SE364, DJ263 and UG273) was compared to the growth 19

of HIV-1Ba-L in each of the tissue explant models. Release of p24 was found to be lower for 20

clade C isolates; UG268 (-0.73 log10 p24, P<0.001) and SE364 (-1.05 log10 p24; P<0.001) 21

compared to HIV-1Ba-L across all explant assays. The clade A isolate (UG273) resulted in lower 22

p24 production compared to HIV-1Ba-L for cervical (-0.24 log10 p24 pg/mL; P<0.05) and tonsil (-23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 17 of 44

0.42 log10 p24 pg/mL; P<0.001) tissues. There was no difference in p24 production between A/G 1

recombinant isolate DJ263 compared to HIV-1Ba-L (P=n.s.) for any of the tissue types tested. 2

Intra-assay variability across experimental conditions. Intra-assay variability was 3

measured using the %CV for all replicate p24 measurements, where a higher %CV indicates 4

greater variability for replicate measurements. The %CVs were compared across assays (Figure 5

6a-d). There was no difference in %CV across experimental conditions (virus/media source, use 6

of PRO 2000 or isolate type; Kruskal Wallis analysis of variance, P=0.26-0.98; Figure 6a-c), 7

with a median %CV across all experimental conditions of 28.8%. Intra-assay reproducibility did 8

vary across explant tissue and model type, where greater variability was found when using the 9

rectal tissue explant method when compared to the total %CV for the study (Mann Whitney Test, 10

P<0.0001; Figure 6d). Tonsil and un-stimulated cervical tissue explant culture resulted in the 11

most reproducible explant virus growth (i.e. median %CV; Figure 6d.). 12

13

Discussion 14

15

While explant cultures are currently used to study HIV pathogenesis and transmission in 16

tissues relevant to in vivo sites of infection, they are also being used to evaluate the antiviral 17

activity of candidate topical microbicides prior to subsequent studies in animals and humans. 18

Given the variety of protocols currently used in tissue explant studies (e.g. (1, 2, 4, 8, 10, 14)), 19

the first aim of this study was to identify a uniform method for analyzing, comparing and 20

presenting virus growth curve data. To this end, the soft endpoint metric provides such a method. 21

SOFT was developed to enable inter-laboratory comparisons of HIV-1 replication across explant 22

methods (cervical, rectal and tonsil), by determining the endpoint to compare virus growth 23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 18 of 44

between control and microbicide-treated tissues. Comparison of virus growth between control 1

and treated tissues at time points prior to the accelerated growth phase, or after virus growth has 2

tapered off, could underestimate the level of virus inhibition exhibited by a microbicide 3

candidate. Comparison of virus growth at SOFT provides an ‘ideal’ endpoint comparison for 4

each growth curve by excluding the variability in measurements at time points prior to SOFT, 5

when the virus control may be at low or non-detectable levels or after SOFT when the system 6

may no longer be able to support viral growth. SOFT aims to provide a sensitive indicator of 7

drug efficacy to be used early in a drug screening program, prior to additional confirmatory 8

testing, where the risks of making a type II error (i.e. false negative, when a potentially 9

efficacious compound is excluded) outweigh the risks of a type I error (i.e. false positive, where 10

a potentially ineffective compound is included). 11

An evaluation of calculation time, ease of performance, availability of resources and 12

other factors that may be important to a laboratory should be considered prior to the adoption of 13

any endpoint method. Although the AUC and SLOPE calculations proved to be effective 14

predictors of virus growth, the complexity of these calculations may reduce their utility for 15

research teams without access to statistical software necessary to compute non-linear regressions 16

and trapezoidal functions. In contrast, SOFT can be readily calculated and thus allows direct 17

comparison of growth curves using standard statistical tests (e.g. ANOVA, t-test, Kruskal-Wallis 18

and Mann Whitney). A multivariate comparison (LDA) of various endpoint approaches further 19

supported the application of SOFT as an objective method for evaluating virus replication. In 20

addition, SOFT neither over nor underestimated virus growth compared to other endpoints and 21

provided a measure of growth that reliably reflected the virus yield of the assay. 22


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 19 of 44

An added advantage to the identification of a robust assay endpoint is the ability to use it 1

for identifying key parameters that, once standardized, can lead to more reproducible data 2

between laboratories. As a second aim of this study, SOFT was used to compare conditions that 3

could affect virus growth in explant assays. The variables found to affect virus growth in the 4

explants studied here included: (i) source/type of assay reagents; (ii) tissue type; (iii) how the 5

tissue was cultured (i.e. tissue stimulation); (iv) HIV-1 isolate used; and (v) microbicide 6

concentration. In contrast, laboratory measurement of p24 protein in culture supernatants was 7

found to be generally consistent, regardless of assay-specific factors such as ELISA 8

manufacturer, standard curve range, method of ELISA analysis, or the laboratory running the 9

analysis. 10

Since HIV-1 replication can be monitored in tissue culture with a variety of methods 11

(including measurement of p24 protein), different patterns of virus growth can be observed 12

depending on whether virus is measured in the cell-free or cell-associated compartments of 13

cultured cells and tissues (3, 25). In the microbicide field, researchers primarily rely on p24 14

protein levels in culture supernatants as a measure of virus replication over the course of the 15

explant study. Using this approach for the current study, greater viral replication was observed in 16

all explant models using the centrally provided, VQA LAB viral stock and the laboratory’s own 17

in-house media formulations. Use of a common virus stock provided by a central repository 18

increased p24 production for all tissue types and methods, but media formulations routinely used 19

by each laboratory were found to be better suited to the laboratory’s preferred explant method. 20

Perhaps the largest effect on p24 production was the tissue type and model used. For 21

example, the lowest viral replication was observed using un-stimulated cervical tissue. When 22

cervical tissue was immune stimulated or co-cultured with PBMC, viral replication was 23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 20 of 44

noticeably increased by 10- or 1000-fold, respectively. Use of either rectal or tonsil tissue also 1

significantly increased viral replication, suggesting that viral replication can be significantly 2

affected by the endogenous stimulation or the concentration and distribution of target cells 3

present in each tissue type or model system. 4

The absolute quantity of p24 protein produced by tissue explant cultures did not appear to 5

correlate with the ability to detect a drug effect. The candidate microbicide PRO 2000 was found 6

to inhibit virus growth for the majority (4/5) of explant methods used by the seven laboratories 7

(i.e. cervical, tonsil, rectal and cervical PBMC co-culture) where p24 production at SOFT ranged 8

from 14-670,300 pg/mL. Cultures releasing both high (tonsil) and low (cervical) p24 levels 9

produced reliable drug effects, while another high p24 release explant model (stimulated 10

cervical) failed to demonstrate any drug effect in this study. 11

Given the lack of a standard endpoint for virus growth in explant studies, few laboratories 12

to date have reported microbicide activity in terms of an IC50. In fact, only one laboratory has 13

published an IC50 for PRO 2000 in cervical tissue as ~80 µg/mL (11). In the current study, the 14

IC50 for PRO 2000 varied widely among the tissue types evaluated (9.7-98.0 og/ml) but 15

remained fairly consistent within an explant method, as demonstrated by the 3 laboratories 16

working with cervical tissue (24.5-29.3og/ml). These data indicate that inhibitory concentrations 17

of PRO 2000 in explant tissues are higher than those in cell lines and primary T cells or 18

macrophages (IC50‘s ranging from 0.7-12.8 µg/mL) (22, 29, 31), suggesting that more drug is 19

required to penetrate tissue in contrast to monolayers or cell suspensions. Comparing the same 20

tissue type used in different model systems, such as cervical and cervical with a PBMC co-21

culture, PRO 2000 was also consistently active (24.5-29.3 og/ml compared to 28.1 og/ml 22

respectively), but not when compared with the stimulated cervical (IC50 could not be determined) 23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 21 of 44

or rectal (9.7og/ml) models. This suggests that drug IC50s could be compared between 1

laboratories using the same tissue type under the same model conditions, but not necessarily 2

between laboratories using either different explant tissues or assay methods. 3

Although the HIV-1 isolate used had a significant impact on virus growth (clade C strains 4

resulted in lower virus growth and clades A and A/G showed comparable growth to HIV-1Ba-L), 5

it is important to note that the explant models examined in this study supported growth of viruses 6

that represent only a sub-sample of isolates available to microbicide researchers (7). These 7

results support the inclusion of non-HIV-1Ba-L isolates in future explant studies by determining 8

microbicide activity against the more clinically relevant, non-laboratory-adapted strains of HIV-9

1. 10

While an intra-assay variability of <20%CV is generally considered acceptable for in 11

vitro assays (26), this study found a wide range for explant intra-assay %CV’s (0.8-163.4%), 12

where rectal tissue measurements were the most variable. High variability in rectal explant 13

tissues may be due to such factors as susceptibility of different tissue areas to infection (immune 14

cell pockets, areas of high M cell populations, etc.) or friability of tissue that is of a less 15

structured type (1, 10). However, even though the high intra-assay variability reduced the 16

statistical power of treatment comparisons, consistent drug and isolate effects were found. 17

Additionally, statistical power was not consistent across explant methods; cervical explants used 18

by three laboratories provided greater statistical power for drug and isolate effects compared to 19

other explant methods tested by only a single laboratory (i.e. cervical stimulated, cervical PBMC 20

co-culture, tonsil and rectal). 21

In summary, in order to improve the quality of data from laboratories using tissue 22

explants to evaluate topical microbicides, this study addressed the following two aims: 1) 23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 22 of 44

identification of a uniform method for analyzing, comparing, and presenting virus growth curve 1

data, and 2) identification of those key parameters that, once standardized, will lead to more 2

reproducible data between laboratories. A comparison of various endpoint approaches 3

demonstrated that the soft endpoint can provide a measure of virus growth that is easily 4

calculated, reliably indicates the virus yield of the assay, and allows direct comparison of growth 5

curves with standard statistical tests. In terms of key assay parameters, use of a common virus 6

stock was shown to be most important for improving virus growth among the tested explant 7

models. In addition, as the data with primary strains indicated, identification of clinically 8

relevant HIV-1 clades that are able to replicate well in tissue explants will be important for future 9

microbicide studies. Although the tissue type and model used had the greatest impact on virus 10

growth, this study also demonstrated that a reliable drug effect can be observed in tissues with 11

low or high p24 protein levels, and that IC50’s can be compared between laboratories using the 12

same tissue type. Finally, wide intra-assay variability may be inherent in tissue explants studies, 13

where the extent of measurement variability found here depended upon the tissue type used. 14

Further studies, using a variety of tissue types, are recommended to determine the number of 15

donors and replicates necessary for adequate statistical power in comparisons of HIV inhibition 16

between compounds. 17

Reproducible tissue explant research has the potential to provide an early indicator of 18

drug efficacy, thus accelerating the best microbicide candidates into subsequent animal studies 19

and, ultimately, human clinical trials. It is hoped that the identification of a reliable endpoint and 20

other key methodological parameters will increase future standardization and comparability of 21

pre-clinical explant assays. 22

23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 23 of 44

ACKNOWLEDGEMENTS 1

2

This work was part of the Microbicide Quality Assurance Program (MQAP) supported by 3

the following contracts from the US National Institute of Health: NICHD N01-HD-3-3350 4

(September 2003-January 2007) and NIAID N01-AI-33350 (February 2007-August 2008). The 5

views of the authors do not necessarily reflect those of the funding agencies. The authors have no 6

conflicts of interest or financial interests regarding the work presented. Investigators using 7

explant assays in their respective microbicide-related research were invited to participate in the 8

design of the studies described in this paper. Technical staff participated in regular conference 9

calls and discussions of the standardized procedures as the studies were implemented and data 10

were acquired. The authors would like to thank the following: Dr. A. Profy, Endo"11

Pharmaceuticals Solutions, Inc. (Lexington, MA) for providing PRO 2000; Dr. Brigitte E. 12

Sanders-Beer (BIOQUAL, Inc., 9600 Medical Center Drive, Rockville, MD ) for her leadership 13

of the MQAP from 2003 thru 2005; Dr. Donald Brambilla (New England Research Institutes, 9 14

Galen Street, Watertown, MA) for helpful discussions at the initial stages of this research, and; 15

Dr. Timothy A. Green (Branch Chief of the Quantitative Sciences and Data Management 16

Branch, Centers for Disease Control and Prevention, GA) for helpful comments on this 17

manuscript. 18

The authors would also like to thank the following NIH Program Staff for their scientific 19

expertise and guidance in relation to the MQAP studies: Drs. Kailash Gupta (NIAID), Fulvia 20

Veronese (Office for AIDS Research to NIAID), and James Turpin (NIAID). The author list 21

includes the Principal Investigators from the participating laboratories and their respective 22

technical representatives. Since participation in the MQAP was voluntary, it should be noted that 23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 24 of 44

the explant studies described in this paper were facilitated by the infrastructure, resources and 1

microbicide related grant support available at each of the following participating laboratories 2

(this order does not correspond to randomly assigned Lab ID letter): 3

1. National Institute of Child Health and Human Development, National Institutes of 4

Health, 10 Center Drive 10/9D58 Bethesda, MD 20892 (NIH Intramural Program). 5

2. Rush University Medical Center, Department of Immunology/Microbiology, 735 6

W. Harrison Street, Room 616 Cohn Chicago, Illinois 60612. [Virology Quality Assurance 7

(VQA) Program supported by NIAID N01-AI-50044]. 8

3. St. George's, University of London, London, SW17 0RE, UK. [DFID/MRD (UK) 9

to RS as part of the Microbicide Development Programme]. 10

4. Magee-Womens Research Institute, University of Pittsburgh, School of Medicine, 11

Department of Obstetrics, Gynecology, and Reproductive Sciences, 204 Craft Avenue, 12

Pittsburgh, PA 15213 (NIH U01 AI068633-07 to CSD). 13

5. Center for Prevention Research at David Geffen School of Medicine, University 14

of California-Los Angeles AIDS Institute [NIH CFAR Mucosal Immunology Core (AI 28997) 15

and NIH IP/CP U19 (AI 060614) to PAA]. 16

17


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 25 of 44

References 1

2

1. Abner, S. R., P. C. Guenthner, J. Guarner, K. A. Hancock, J. E. Cummins, Jr., A. 3

Fink, G. T. Gilmore, C. Staley, A. Ward, O. Ali, S. Binderow, S. Cohen, L. A. 4

Grohskopf, L. Paxton, C. E. Hart, and C. S. Dezzutti. 2005. A human colorectal 5

explant culture to evaluate topical microbicides for the prevention of HIV infection. J 6

Infect Dis 192:1545-56. 7

2. Collins, K. B., B. K. Patterson, G. J. Naus, D. V. Landers, and P. Gupta. 2000. 8

Development of an in vitro organ culture model to study transmission of HIV-1 in the 9

female genital tract. Nat Med 6:475-9. 10

3. Collman, R., N. F. Hassan, R. Walker, B. Godfrey, J. Cutilli, J. C. Hastings, H. 11

Friedman, S. D. Douglas, and N. Nathanson. 1989. Infection of monocyte-derived 12

macrophages with human immunodeficiency virus type 1 (HIV-1). Monocyte-tropic and 13

lymphocyte-tropic strains of HIV-1 show distinctive patterns of replication in a panel of 14

cell types. J Exp Med 170:1149-63. 15

4. Cummins, J. E., Jr., J. Guarner, L. Flowers, P. C. Guenthner, J. Bartlett, T. 16

Morken, L. A. Grohskopf, L. Paxton, and C. S. Dezzutti. 2007. Preclinical testing of 17

candidate topical microbicides for anti-human immunodeficiency virus type 1 activity 18

and tissue toxicity in a human cervical explant culture. Antimicrob Agents Chemother 19

51:1770-9. 20

5. Cummins, J. E., N. Richardson-Harman, J. Bremer, P. Anton, C. Dezzutti, P. Gupta, N. 21

Lurain, L. Margolis, R. Shattock, and P. Reichelderfer. 2006. Multi-Center Evaluation of 22


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 26 of 44

Explant Models for Topical Microbicide Development. Presented at the 2006 Conference 1

on Retroviruses and Opportunistic Infections, Denver, Colorado. 2

6. Dezzutti, C. S., V. N. James, A. Ramos, S. T. Sullivan, A. Siddig, T. J. Bush, L. A. 3

Grohskopf, L. Paxton, S. Subbarao, and C. E. Hart. 2004. In vitro comparison of 4

topical microbicides for prevention of human immunodeficiency virus type 1 5

transmission. Antimicrob Agents Chemother 48:3834-44. 6

7. Finzi, D., and O. K. Sharma October 13 2008, posting date. The Source of Critical HIV 7

Research Materials https://www.aidsreagent.org/Index.cfm. [Online.] 8

8. Fischetti, L., S. M. Barry, T. J. Hope, and R. J. Shattock. 2008. HIV-1 infection of 9

human penile explant tissue and protection by candidate microbicides. AIDS. 10

9. Fletcher, P., Y. Kiselyeva, G. Wallace, J. Romano, G. Griffin, L. Margolis, and R. 11

Shattock. 2005. The nonnucleoside reverse transcriptase inhibitor UC-781 inhibits 12

human immunodeficiency virus type 1 infection of human cervical tissue and 13

dissemination by migratory cells. J Virol 79:11179-86. 14

10. Fletcher, P. S., J. Elliott, J. C. Grivel, L. Margolis, P. Anton, I. McGowan, and R. J. 15

Shattock. 2006. Ex vivo culture of human colorectal tissue for the evaluation of 16

candidate microbicides. AIDS 20:1237-45. 17

11. Fletcher, P. S., G. S. Wallace, P. M. Mesquita, and R. J. Shattock. 2006. Candidate 18

polyanion microbicides inhibit HIV-1 infection and dissemination pathways in human 19

cervical explants. Retrovirology 3:46. 20

12. Flint, S. J., L. W. Enquist, V. R. Racaniello, and A. M. Skalka (ed.). 2004. Principles 21

of Virology: Molecular Biology, Pathogenesis, and Control of Animal Viruses, 2nd Ed. 22


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 27 of 44

13. Fox-Canale, A. M., T. J. Hope, J. Martinson, J. R. Lurain, A. W. Rademaker, J. W. 1

Bremer, A. Landay, G. T. Spear, and N. S. Lurain. 2007. Human cytomegalovirus and 2

human immunodeficiency virus type-1 co-infection in human cervical tissue. Virology 3

369:55-68. 4

14. Glushakova, S., B. Baibakov, L. B. Margolis, and J. Zimmerberg. 1995. Infection of 5

human tonsil histocultures: a model for HIV pathogenesis. Nat Med 1:1320-2. 6

15. Greenhead, P., P. Hayes, P. S. Watts, K. G. Laing, G. E. Griffin, and R. J. Shattock. 7

2000. Parameters of human immunodeficiency virus infection of human cervical tissue 8

and inhibition by vaginal virucides. J Virol 74:5577-86. 9

16. Grivel, J. C., J. Elliott, A. Lisco, A. Biancotto, C. Condack, R. J. Shattock, I. 10

McGowan, L. Margolis, and P. Anton. 2007. HIV-1 pathogenesis differs in 11

rectosigmoid and tonsillar tissues infected ex vivo with CCR5- and CXCR4-tropic HIV-12

1. AIDS 21:1263-72. 13

17. Grivel, J. C., and L. B. Margolis. 1999. CCR5- and CXCR4-tropic HIV-1 are equally 14

cytopathic for their T-cell targets in human lymphoid tissue. Nat Med 5:344-6. 15

18. Gupta, P., K. B. Collins, D. Ratner, S. Watkins, G. J. Naus, D. V. Landers, and B. K. 16

Patterson. 2002. Memory CD4(+) T cells are the earliest detectable human 17

immunodeficiency virus type 1 (HIV-1)-infected cells in the female genital mucosal 18

tissue during HIV-1 transmission in an organ culture system. J Virol 76:9868-76. 19

19. Gupta, P., D. Ratner, B. K. Patterson, K. Kulka, L. C. Rohan, M. A. Parniak, C. E. 20

Isaacs, and S. Hillier. 2006. Use of frozen-thawed cervical tissues in the organ culture 21

system to measure anti-HIV activities of candidate microbicides. AIDS Res Hum 22

Retroviruses 22:419-24. 23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 28 of 44

20. Khattree, R. 2000. Multivariate Data Reduction and Discrimination with SAS ® 1

Software. SAS Institute Inc. , Cary, NC. 2

21. Klasse, P. J., R. J. Shattock, and J. P. Moore. 2006. Which topical microbicides for 3

blocking HIV-1 transmission will work in the real world? PLoS Med 3:e351. 4

22. Lackman-Smith, C., C. Osterling, K. Luckenbaugh, M. Mankowski, B. Snyder, G. 5

Lewis, J. Paull, A. Profy, R. G. Ptak, R. W. Buckheit, Jr., K. M. Watson, J. E. 6

Cummins, Jr., and B. E. Sanders-Beer. 2008. Development of a comprehensive human 7

immunodeficiency virus type 1 screening algorithm for discovery and preclinical testing 8

of topical microbicides. Antimicrob Agents Chemother 52:1768-81. 9

23. Munoz-Fernandez, M. A., J. Navarro, M. G. Montes, J. Cosin, J. M. Zabay, and E. 10

Fernandez-Cruz. 1996. Quantification of low levels of human immunodeficiency virus 11

(HIV) type 1 RNA in P24 antigen-negative, asymptomatic, HIV-positive patients by 12

PCR. J Clin Microbiol 34:404-8. 13

24. Niioka, T., T. Uno, N. Yasui-Furukori, M. Shimizu, K. Sugawara, and T. Tateishi. 14

2006. Identification of the time-point which gives a plasma rabeprazole concentration 15

that adequately reflects the area under the concentration-time curve. Eur J Clin 16

Pharmacol 62:855-61. 17

25. Patterson, B. K., A. Landay, J. N. Siegel, Z. Flener, D. Pessis, A. Chaviano, and R. 18

C. Bailey. 2002. Susceptibility to human immunodeficiency virus-1 infection of human 19

foreskin and cervical tissue grown in explant culture. Am J Pathol 161:867-73. 20

26. Reed, G. F., F. Lynn, and B. D. Meade. 2002. Use of coefficient of variation in 21

assessing variability of quantitative assays. Clin Diagn Lab Immunol 9:1235-9. 22


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 29 of 44

27. Richardson, B. A., and J. Overbaugh. 2005. Basic statistical considerations in 1

virological experiments. J Virol 79:669-76. 2

28. Roth, S., M. Monsour, A. Dowland, P. C. Guenthner, K. Hancock, C. Y. Ou, and C. 3

S. Dezzutti. 2007. Effect of topical microbicides on infectious human immunodeficiency 4

virus type 1 binding to epithelial cells. Antimicrob Agents Chemother 51:1972-8. 5

29. Rusconi, S., M. Moonis, D. P. Merrill, P. V. Pallai, E. A. Neidhardt, S. K. Singh, K. 6

J. Willis, M. S. Osburne, A. T. Profy, J. C. Jenson, and M. S. Hirsch. 1996. 7

Naphthalene sulfonate polymers with CD4-blocking and anti-human immunodeficiency 8

virus type 1 activities. Antimicrob Agents Chemother 40:234-6. 9

30. SAS, Institute, and Inc. 1990. SAS/STAT, Users Guide, Version 6, 4th Edition. SAS 10

Institute Inc. , Cary, NC. 11

31. Scordi-Bello, I. A., A. Mosoian, C. He, Y. Chen, Y. Cheng, G. A. Jarvis, M. J. Keller, 12

K. Hogarty, D. P. Waller, A. T. Profy, B. C. Herold, and M. E. Klotman. 2005. 13

Candidate sulfonated and sulfated topical microbicides: comparison of anti-human 14

immunodeficiency virus activities and mechanisms of action. Antimicrob Agents 15

Chemother 49:3607-15. 16

32. Senneke, S., Richardson-Harman, N., Cummins, J., Lackman-Smith, C., Bromley, 17

C. & Reichelderfer, P. 2006. A Soft Endpoint for HIV-1Ba-l Growth in Cervical, 18

Rectal, and Tonsular In Vitro Assays. Presented at the 2006 Joint Statistical Meetings, 19

Seattle, Washington. 20

33. Sutthent, R., N. Gaudart, K. Chokpaibulkit, N. Tanliang, C. Kanoksinsombath, and 21

P. Chaisilwatana. 2003. p24 Antigen detection assay modified with a booster step for 22


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 30 of 44

diagnosis and monitoring of human immunodeficiency virus type 1 infection. J Clin 1

Microbiol 41:1016-22. 2

34. Taylor, R. N., K. M. Fulford, and A. Y. Huong. 1978. Comparison of kinetic and end-3

point diffusion methods for quantitating human serum immunoglobulins. J Clin 4

Microbiol 8:23-7. 5

35. Tsai, W. P., S. R. Conley, H. F. Kung, R. R. Garrity, and P. L. Nara. 1996. 6

Preliminary in vitro growth cycle and transmission studies of HIV-1 in an autologous 7

primary cell assay of blood-derived macrophages and peripheral blood mononuclear 8

cells. Virology 226:205-16. 9

36. Wang, G. P., and F. D. Bushman. 2006. A statistical method for comparing viral 10

growth curves. J Virol Methods 135:118-23. 11

37. Yeh, S. H. 1991. Using the trapezoidal rule for the area under the curve calculation. 12

SAS®

Users Group International. SAS®

Users Group International. 27: 227-229. . 13

14

15

SAS®

is a registered trade mark of SAS Institute Inc., in the USA and other countries. 16

® Indicates USA registration of the SAS Institute Inc., Cary, NC, USA. 17

18

19


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 31 of 44

Figure Captions 1

2

3

FIGURE 1. Soft endpoint determination of HIV-1Ba-L growth in VQAB/IHM tissue 4

explants. The SOFT endpoint (?) is shown for: (a) low (MAXp24 < 1500 pg/ml); (b) medium 5

(MAXp24 1500 - 20,000 pg/ml); and (c) high (MAXp24 > 20,000 pg/ml) yielding assays. Each 6

line represents virus growth for each of 12 donors, where each donor is distinguished by the line 7

pattern within each panel. 8

9

FIGURE 2. A comparison of five endpoint measures to determine HIV-1Ba-L growth 10

in explant tissues. Viral growth determined at the endpoints SOFT, DAY12, DAY15 and AUC 11

are plotted on the left y axis (log10 p24 pg/ml) whilst the SLOPE is plotted on the right y axis for 12

low (?), medium (») and high (?) yielding assays. Horizontal lines indicate the median of each 13

endpoint. Note that for two of the high growth assays (Laboratory D; donors 1 and 2), growth 14

was only measured for 12 days. 15

16

FIGURE 3. The effect of explant method type on virus yield. Virus growth (p24) was 17

compared across explant methods by Kruskal Wallis analysis of variance (P<0.0001). The p24 at 18

the assay soft endpoint is presented for HIV-1Ba-L grown in cervical (14 donors), stimulated 19

cervical (6 donors), cervical with PBMC co-culture (2 donors), tonsil (4 donors) and rectal (4 20

donors) explant experiments, sourced from IHB, IHM, VQAB and VQAM conditions. Median 21

and inter-quartile ranges are indicated for each method. The table shows the probability values 22


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 32 of 44

for significantly different pair wise comparisons (Dunn’s multiple comparison test). n.s. = not 1

significant. 2

3

FIGURE 4. Application of SOFT analyses to “low” viral growth in un-stimulated 4

cervical tissue for donors 1 (a), 2 (b) and 3 (c) and % virus inhibition across donors (d). 5

Data shown represents the viral growth in the absence (», solid lines) or presence (dotted lines) 6

of the candidate microbicide, PRO-2000 ( 5 og/ml, 50 og/ml and 500 og/ml). Medians 7

and inter-quartile ranges for replicate measurements are shown. The SOFT endpoint (ï) for 8

donors 1 and 2 occurred at day 10 whilst for donor 3 occurred at day 14. Inhibition of viral 9

infection determined using different endpoint analyses (d). The % virus inhibition results were 10

compared for PRO-2000 (5, 50 and 500og/ml) for three donor cervical tissue explant tissue 11

samples from one laboratory. Virus inhibition (%) was calculated using p24 measurements 12

using: (i) SOFT; (ii) DAY10; and (iii) DAY14 endpoint analysis. The median and inter-quartile 13

ranges across replicates and donors are shown. Virus inhibition for each treatment was compared 14

to BaL control at SOFT, DAY12 and DAY14 (Wilcoxon Rank Signed Test; *P<0.05, 15

**P<0.01). 16

17

FIGURE 5. Inhibition of HIV-1Ba-L infection by PRO 2000. Inhibition of viral 18

infection by PRO 2000 was determined for five explant tissue types (cervical, stimulated 19

cervical, cervical with PBMC co-culture, tonsil and rectal) in seven laboratories (A-G). (a) 20

Median % virus control values were compared across three concentrations of PRO 2000 21

( 5og/ml, 50og/ml and 500og/ml) to 100% virus control, where significant effects of 22

PRO 2000 are indicated by * P<0.05, ** P<0.01 and *** P< 0.001 (Wilcoxon Rank Signed test). 23


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 33 of 44

Data represent the median and inter-quartile range of % virus control for each tissue type. (b) 1

The half maximal (50%) inhibitory concentration (IC50) for PRO 2000 was calculated using p24 2

released at the assay soft endpoint and derived by non-linear regression analysis for each tissue 3

type: stimulated cervical ( ), cervical with PBMC co-culture ( ), rectal ( ), and 4

tonsil( ). (c) The IC50 for PRO 2000 was calculated using p24 released at the assay soft 5

endpoint and derived by non-linear regression analysis for each laboratory using un-stimulated 6

cervical tissue: Labs A ( ), B ( ) and E ( ). Data represent the replicate % virus 7

control for each lab. The embedded tables (Figure 5 b & c) outline the individual IC50 (たg/ml) 8

values, upper and lower 95% confidence intervals and the goodness of fit (R2) of each curve to 9

the % virus control data, n/a indicates where values could not be estimated with confidence from 10

the non-linear regression analysis. 11

12

FIGURE 6. Intra-assay variability across experimental conditions. Intra-assay 13

variability was measured using the %CV for replicate p24 measurements. Data shown represent 14

the median %CV and inter-quartile range for p24 release determined at the assay soft endpoint 15

for seven laboratories using their preferred explant method in each experimental condition 16

alongside the total %CV for the study . (a) Source of virus/media; (b) Effect of microbicide 17

inhibitor PRO 2000; (c) Growth of different HIV-1 isolates; and (d) Tissue type. Significant 18

differences were determined using the Mann Whitney test. 19


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 34 of 44

Figure 1 1

Day 0 Day 3 Day 6 Day 9 Day 12 Day 150

500

1000

1500

p2

4 (

pg

/ml)


5000

10000

15000

p2

4 (

pg

/ml)


50000

100000

150000

200000

p2

4 (

pg

/ml)

Days Post Washout

a.

b.

c.

2


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 35 of 44

1

2

3

4

Figure 2 5

6

AUC SOFT DAY12 DAY15 SLOPE

0

5

10

15

1

10

100

1000

Assay Endpoint

p2

4 (

log

10 p

g/m

l)

Slo

pe

7

8

9

10

11

12


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 36 of 44

Figure 3 1

2

1

10

100

1000

10000

100000

1000000

Explant Method

(no. of labs)

Cervical

(3)

Cervical

Stimulated

(1)

Tonsil

(1)

Rectal

(1)

Cervical

PBMC

Co-culture

(1)

Explant Method Cervical

Stimulated

Cervical

PBMC

Co-culture

Tonsil Rectal

Cervical <0.001 <0.001 <0.001 <0.05

Cervical Stimulated ~ <0.001 n.s. n.s.

Cervical PBMC Co-culture ~ ~ n.s. <0.01

Tonsil ~ ~ ~ <0.05

p2

4 (

pg/m

l)

3


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 37 of 44

1

Figure 4 2

0 2 4 6 8 10 12 140

100

200

300

SOFT

p2

4 (

pg/m

l)

0 2 4 6 8 10 12 140

50

100

150

200

250

SOFT

p2

4 (

pg/m

l)

0 2 4 6 8 10 12 140

50

100

150

200

250

SOFT

p2

4 (

pg/m

l)

a. Donor 1

b. Donor 2

c. Donor 3

Day post washout

3


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 38 of 44

Figure 4 d.

5 50 500 5 50 500 5 50 5000

20

40

60

80

100

SOFT Day 10 Day 14

* ***

PRO 2000 (og/ml)

Endpoint

% V

irus

Inhib

itio

n


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 39 of 44

Figure 5

a.

0

20

40

60

80

100

A B E C G F D

CervicalCervical

Stimulated

Cervical

PBMC

Co-culture

Rectal Tonsil

LabExplant

Method

***

*** ***

***

***

*** ***

**

***

***

**

***

% V

iru

s C

on

tro

l


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 40 of 44

Figure 5b.

0.5 1.0 1.5 2.0 2.5 3.0

0

25

50

75

100

125

95% CI Explant Method

IC50

(たg/ml) Lower Upper R

2

Tonsil 9.7 6.9 13.8 0.81

Cervical PBMC Co-Culture 28.1 9.3 84.7 0.78

Rectal 98.0 38.6 248.9 0.45

Cervical Stimulated n/a n/a n/a -0.28

Log10 [PRO 2000] og/ml

% V

iru

s C

on

tro

l


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 41 of 44

Figure 5c.

0.5 1.0 1.5 2.0 2.5 3.00

50

100

150

95% CI Cervical Un-stimulated

IC50

(たg/ml) Lower Upper R

2

Lab A 25.4 5.9 110.3 0.24

Lab B 24.5 7.5 80.2 0.28

Lab E 29.3 11.7 73.4 0.59

All Cervical Labs 21.8 9.6 49.2 0.28

Log10 [PRO 2000] og/ml

% V

irus

Co

ntr

ol


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 42 of 44

Figure 6

IHB/IH

M

IHB/V

QA

M

VQ

AB/IH

M

VQ

AB/V

QA

M

Total

%CV

0

50

100

150

200

% C

V

5PRO

50PR

O

500P

RO

Total

%CV

0

50

100

150

200

% C

V

DJ2

63

SE364

UG

268

UG

273

Ba-

L

Total

%CV

0

50

100

150

200

% C

V

Cer

vica

l

Cer

vica

l Stim

ulat

ed

Cer

vica

l PBM

C C

o-Cul

ture

Tonsil

Rec

tal

Total

%CV

0

50

100

150

200

P<0.0001

% C

V

a. b.

c. d.


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 44 of 45

Table 1. Description of explant media and p24 methods used for individual laboratories and the VQA LAB.

Lab Code

(reference) Tissue Type Polarized

Tissue

Size

(mm*)

Base

Medium

Serum or

Additives Antibiotics p24 Kit

a VQA LAB n/a n/a n/a R 20% FBS, GL Gent, Amp Beckman Coulter

A (11) Cervical no 3x3x2 R 10% FBS, GL PS Beckman Coulter

B (4) Cervical no 3x3x2 D 10% HuAB,

GL PS Perkin Elmer

C (1) Stimulated

Cervical yes 5x5x3 D

10% HuAB,

100 U/ml IL-2 PS

bNCI

D (17) Tonsil no 2x2x2 R 15% FBS, GL,

N Gent; F

Beckman Coulter

(kinetic)

E (13) Cervical no 3x3x3 R 20% FBS, GL Gent, Amp Zeptometrix

F (10) Rectal no 3x3x2 R 10% FBS, GL Z; F bNCI

G (2, 18, 19)§ Cervical

(PBMC) yes 5x5x3 R 20% FBS, GL PS Perkin Elmer

Media: R=RPMI 1640 = Roswell Park Memorial Institute Media; D=DMEM = Dulbecco's Modified Eagle Medium; Additives: GL=2-4mM L-glutamine; huAB

= human AB serum; N=1X NEAA = non-essential amino acids (100x); FBS = Fetal bovine serum; HuAB = human AB serum; Antibiotics: Gent=50たg/ml

Gentamicin sulfate, Amp = 50たg/ml Ampicillin, PS = 100U/ml Penicillin 100たg/ml Streptomycin, Z = 50g/ml Zosyn, F = Fungizone (amphotericin B); PBMC =

peripheral blood mononuclear cell co-culture. a VQA LAB analyzed matched culture supernatants for p24 measurements on all laboratories and provided the VQA media (VQAM). b AIDS, Vaccine Program, NCI-Frederick Cancer Research and Development Center HIV-1 p-24 Antigen Capture Assay. §Laboratory G followed the published method with the following modifications: HIV-1 transmission across the mucosa was determined by measuring HIV-1 p24

antigen (p24) in supernatants collected from PBMC cultures maintained in the bottom chamber of a transwell system.

* presented as length x width x height.


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/


Page 45 of 45

TABLE 2. Linear discriminant analysis of the various endpoint measures.

Endpoint measure of virus growth R2 F Value Probability (F)

SOFT 0.93 57.24 <0.0001

LogSOFT* 0.96 140.02 <0.0001

DAY12 0.67 9.12 0.007

DAY15 0.38 2.71 0.120

AUC 0.60 6.76 0.016

SLOPE 0.68 9.51 0.006

Results of the LDA where the virus endpoint measures of viral growth (pg/ml p24) in explant tissues differentiated

between low (5 assays), medium (3 assays) and high (4 assays). *Due to multico-linearity between the linear and

logarithmic soft endpoint results, the Log10 p24 at the soft endpoint (LogSOFT) was entered into a separate LDA.


http://jcm.asm

.org/D

ownloaded from

http://jcm.asm.org/

Date post:	24-Mar-2018
Category:	Documents
Upload:	truongdang
View:	213 times
Download:	1 times

JCM Accepts, published online ahead of print on 2 September...

Documents