NATAP: Transmitted Resistant HIV- More Serious

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Progressive reversion of HIV-1 mutations may have serious consequences

By Rauscher

NEW YORK (Reuters Health) - Although multidrug-resistant HIV strains, because

they are potentially less virally fit than non-drug resistant strains, were

hoped to be less likely to cause disease, a report published this month

suggests just the opposite.

After transmission of a resistant HIV strain, the virus is able to evolve to

become more fit and cause significant disease, according to the study in the

December 15th issue of Clinical Infectious Diseases.

" The frequency of HIV that is resistant to current medications seems to be

growing, and this may limit global treatment strategies, " Dr. Rajesh T. Gandhi

from Partners AIDS Research Center in Boston, who led the study, noted in

comments to Reuters Health.

Dr. Gandhi and colleagues monitored viral evolution after transmission of

HIV-1 containing multiple reverse transcriptase (RT) and protease mutations (PR)

in a 32-year-old previously healthy man.

In the absence of ongoing antiretroviral therapy, 5 of 12 drug resistance

mutations reverted in a stepwise fashion to wild type over the course of one

year. Of note, reversal of the M184V mutation alone did not produce a change in

replicative capacity but it did enhance resistance to zidovudine and tenofovir.

However, " reversions of a second (RT) mutation and 3 PR mutations were

associated with an increase in viral replicative capacity and this was

temporally

correlated with a marked decrease in CD4 cell number, " the researchers report.

Dr. Gandhi and colleagues say this study " demonstrates the gradual stepwise

back-mutation of certain drug resistance mutations in vivo in the absence of

ongoing drug selection pressure. "

While larger studies are needed to confirm these findings, Dr. Gandhi told

Reuters Health, this report " emphasizes that the recent increase in HIV

transmission among men who have sex with men in the United States may result in

acquisition of multidrug resistant virus, with very serious consequences. "

Clin Infect Dis 2003;37:1693-1698.

ORIGINAL ARTICLE EXCERPTS

“Progressive Reversion of Human Immunodeficiency Virus Type 1 Resistance

Mutations In Vivo after Transmission of a Multiply Drug-Resistant Virusâ€

Rajesh T. Gandhi,1,2,3 Alysse Wurcel,1,2,3 S. Rosenberg,1,2,3 N.

ston,1,2,3 Hellmann,4,a Bates,4 S. Hirsch,1,2,3 and

Bruce D. 1,2,3

1Infectious Diseases Unit and 2Partners AIDS Research Center, Massachusetts

General Hospital, and 3Division of AIDS, Harvard Medical School, Boston; and

4ViroLogic, South San Francisco, California

ABSTRACT

Evolution and transmission of multiply drug-resistant human immunodeficiency

virus type 1 (HIV-1) may limit therapeutic options as global treatment efforts

expand. However, the stability of these mutants in the absence of drug

selection pressure is not known.

We performed a longitudinal analysis of plasma virus from a person who

acquired HIV-1 that contained multiple reverse transcriptase (RT) and protease

(PR)

mutations. In the absence of therapy, 5 of 12 drug resistance mutations

reverted in a stepwise fashion to wild type over the course of 52 weeks.

Reversion

of the M184V mutation alone did not change viral replicative capacity (RC), but

it led to enhanced resistance to zidovudine and tenofovir.

However, reversions of a second RT mutation and 3 PR mutations were

associated with an increase in viral RC, and this was temporally correlated with

a

marked decrease in CD4 cell number.

This study demonstrates the gradual stepwise back-mutation of certain drug

resistance mutations in vivo in the absence of ongoing drug selection pressure.

Moreover, it suggests that, despite initially impaired viral fitness, a

transmitted HIV-1 isolate with multiple drug resistance mutations can evolve to

develop increased RC and significant pathogenicity.

BACKGROUND

Combination antiretroviral therapy (ART) has substantially decreased the

number of deaths resulting from AIDS in the United States and Europe, and its

use

is now being extended to resource-poor settings. There is concern that

evolution and transmission of multiply drug-resistant HIV-1 may limit future

therapeutic options. However, the stability of these mutants in the absence of

the

drug selection pressure under which they were generated is not known. In

addition, limited information is available about the natural history of

infection with

multiply drug-resistant HIV-1. Because resistance mutations can impair viral

fitness, this may affect the rate of progression to AIDS.

Several longitudinal studies that have included patients with chronic HIV-1

infection describe the changes in drug resistance mutations that occur after

incompletely suppressive ART regimens are discontinued. For example, in patients

who have persistent viremia and drug-resistant virus during receipt of a

protease inhibitor (PI)containing regimen, the circulating virus generally

becomes

susceptible to PIs within 12 weeks after all antiretroviral medication is

stopped, which suggests that an outgrowth of wild-type virus occurs once drug

selection pressure is removed. In such patients, the in vitro replicative

capacity (RC) of drug-resistant virus is often low while the patient is

receiving a

failing treatment regimen; however, after discontinuation of therapy, the viral

RC increases coincident with the outgrowth of wild-type virus. Whether the

same pattern occurs after transmission of resistant virus is not certain,

although there is recent evidence that detectable resistance may persist longer

under these conditions.

In this report, we present a detailed longitudinal analysis of viral

evolution after transmission of HIV-1 containing multiple genotypic and

phenotypic

mutations in both reverse transcriptase (RT) and protease (PR). In the absence

of

ongoing ART, virus containing multiple mutations underwent stepwise reversion

to wild type, demonstrating the intrinsic instability of certain drug

resistance mutations in vivo. The progressive reversion of mutations was

associated

with increased viral RC and accelerated CD4 cell count decline. This suggests

that, despite initially reduced viral fitness, a multiply drug-resistant HIV-1

isolate can evolve to develop increased RC and significant pathogenicity.

CASE REPORT

A 32-year-old previously healthy man presented with fever and sore throat 3

weeks after participating in unprotected receptive anal intercourse with an

anonymous male partner. A pharyngeal culture was positive for group A

streptococcus, and the patient received a 10-day course of oral penicillin. In

addition,

the patient was HIV-1 antibody negative, and his HIV-1 RNA level was >750,000

copies/mL, which is consistent with acute HIV-1 infection. His CD4 cell count

was 399 cells/mm3.

The patient was enrolled in an institutional review board approved study of

acute HIV-1 infection at Massachusetts General Hospital (Boston). A viral

isolate was sent to the laboratory for genotyping (Virologic; South San

Francisco,

CA), and an ART regimen that included zidovudine, lamivudine, abacavir, and

efavirenz was initiated 2 weeks after the diagnosis of HIV-1 infection was made

(5 weeks after the unprotected sexual contact). Treatment was discontinued

when the results of genotyping revealed that the patient had been infected with

a

highly resistant isolate of HIV-1. The total duration of the initial

therapeutic regimen was 3 weeks. On the day that treatment was discontinued, the

patient's plasma HIV-1 RNA level was 12,400 copies/mL. Another isolate was

genotyped on the day of treatment discontinuation and did not demonstrate any

new drug

resistance mutations.

Subsequently, the patient's plasma HIV-1 RNA levels and CD4 cell counts were

measured every 13 months; during that time, the patient did not receive ART.

After discontinuation of therapy, his plasma HIV-1 RNA level increased to

>100,000 copies/mL, and his CD4 cell count initially stabilized between 300 and

490

cells/mm3.

Approximately 40 weeks after the initial infection, the patient's CD4 cell

count began to decrease precipitously. Over the course of an 8-week period, his

CD4 cell count decreased from 302 to 73 cells/mm3. A regimen that included

tenofovir, lamivudine, stavudine, didanosine, and efavirenz was initiated at

week

52 after infection. The patient's plasma HIV-1 RNA level decreased from

257,000 to <50 copies/mL and his CD4 cell count increased to 203 cells/mm3

within

24 weeks after initiation of this antiretroviral regimen.

METHODS

Plasma HIV-1 RNA levels were quantified using an RT-PCR method (Amplicor

HIV-1 Monitor assay; Roche Diagnostics). Population-based nucleotide sequencing

of

HIV-1 pol sequences (GeneSeq HIV genotyping assay) was performed by

ViroLogic. Cryopreserved plasma samples were also analyzed for drug

susceptibility

(phenotyping) by a recombinant virus assay (PhenoSense HIV; ViroLogic). The

relative RC of the subject's virus was determined by a modification of the drug

susceptibility assay. HIV-1 RT and PR sequences from clinical specimens were

cloned into recombinant test vectors containing a luciferase indicator gene that

allows for quantification of viral replication. The replication of vectors

containing patient-derived pol sequences was compared with that of a vector

containing wild-type sequence (based on the reference strain HIV NL4-3) in the

absence of antiretroviral drug. The RC was calculated as the ratio of luciferase

activity from vectors containing patient-derived viral sequences to the activity

from the NL4-3 wild-type reference vector. The RC measurement of the

patient-derived virus is expressed as a percentage of NL4-3 RC, which is set at

100%. A

percentage <100% indicates that the patient's HIV-1 strain has a reduced RC,

compared with that of NL4-3. The median RC of wild-type clinical isolates in

this assay is about 60%.

RESULTS

Initial resistance testing. The initial HIV-1 genotyping performed at the

time of diagnosis demonstrated multiple drug resistance mutations in the

viral RT (M41L, M184V, and T215Y) and PR (L10F, L33F, M36I, I54V, A71V, V82A,

I84V, and L90M). Phenotypic drug susceptibility testing confirmed high-level

resistance to lamivudine and zidovudine. In addition, there was at least 23-fold

decreased susceptibility of the plasma virus to all approved PIs, including

lopinavir.

Longitudinal assessment of viral genotype. The subject underwent

longitudinal monitoring of viral genotype while he was not receiving ART. Serial

viral

genotyping revealed stepwise loss of mutations in RT. The first reversion was

noted at day 126, with the emergence of a mixture of wild-type and mutant

virus at codon 184 in RT, which mediates resistance to lamivudine. By day 196,

the entire population of detectable virus in plasma encoded the wild-type

methionine at this position. On day 301, we first detected a mixture of tyrosine

and

cysteine at codon 215. By day 343, all detectable plasma virus encoded

cysteine at position 215. Thus, 2 of 3 RT mutations reverted over the course of

1

year in the absence of therapy.

Similar genotypic analysis of PR mutations likewise demonstrated gradual

detection of mixtures that first began to appear at day 301, when the L10F

mutant

became a mixture. By day 343, 3 of the 9 PR mutations contained mixtures that

included wild-type amino acids (L10L/F/V, I54I/V, and V82V/A).

Phenotypic evolution in the absence of drug selection pressure. To assess

the impact of these reversions on drug susceptibility, longitudinal

phenotypic resistance testing was performed. As resistance to lamivudine waned

(from

>105-fold decreased susceptibility at day 28 to 3.7-fold decreased

susceptibility at day 175), resistance to zidovudine increased (from 19- to

53-fold

decreased susceptibility). An increase in resistance to tenofovir was also seen

as

lamivudine resistance decreased. Reversion at position 215 was associated with a

dramatic reduction in phenotypic resistance to zidovudine. Despite the

appearance of mixtures of wild-type and mutant amino acids in 3 of 9 PR mutation

sites, the virus continued to demonstrate high-level phenotypic resistance to

PIs.

RC(replication capacity). The in vitro RC of the patient's viral isolate

was initially low (9%20% of wild type). Between day 301 and day 364 after

infection, the RC of the circulating virus progressively increased, to 89% of

wild

type. This increase in RC paralleled the reversion of T215Y to T215C in RT

and the emergence of mixtures at 3 PR codons (L10L/F/V, I54I/V, and V82V/A). In

addition, there was a close temporal relationship between the increase in RC

and the rapid decrease in the patient's CD4 cell count.

DISCUSSION

This report demonstrates transmission of an HIV-1 isolate with high-level

resistance to multiple antiretroviral agents, including lopinavir, and documents

longitudinally the changes in genotypic and phenotypic resistance in the

absence of antiretroviral drug selection pressure. The patient infected with

this

virus suffered progressive immunodeficiency within a short time after

infection, and a temporal correlation was seen between the rapid decrease in CD4

cell

count, the increase in viral RC, and the reversion of specific mutations in

viral RT and PR.

Several studies have suggested that transmission of drug-resistant HIV-1 is

increasing in frequency. To date, transmission of virus that is resistant to

lopinavir has not been documented. However, given that drug resistance to

lopinavir in chronically infected individuals has been seen, it is not

surprising

that transmission of such virus can occur. Because of the increasing frequency

of transmission of resistant virus, it has been suggested that resistance t

esting should be performed on isolates from all patients thought to have recent

infection with HIV-1. The present report supports such an approach.

Once it was clear that the patient had been infected with a highly resistant

virus, we discontinued ART. Although early therapy may be associated with

preservation of important HIV-specific immune responses, we felt that this

theoretical benefit was outweighed by the likelihood that selection for further

drug

resistance would occur, because options for a fully suppressive regimen were

limited (tenofovir was not available when the patient was initially evaluated).

Thus, we were able to longitudinally monitor changes in drug resistance

genotype and phenotype in the absence of drug selection pressure. We found that

this individual's plasma virus underwent slow stepwise reversion of mutations in

RT and PR, rather than rapid outgrowth of wild-type virus.

This pattern of stepwise reversion of PR mutations contrasts with what is

generally seen after discontinuation of ART in individuals chronically infected

with HIV-1 for whom this class of therapy is failing. Such patients generally

have outgrowth of previously latent wild-type virus within 48 weeks after

discontinuation of the PI-containing regimen. This observation is consistent

with

out-competition of resistant virus by wild-type virus when drug selection

pressure is removed, perhaps because the wild-type virus is more replication

competent. One likely explanation for the different pattern seen in our patient

is

that he may have been infected only with resistant virus, rather than harboring

a mixture of resistant and wild-type virus, as is the case for individuals

who develop resistance during treatment. The persistence of resistance mutations

in the absence of drug selection pressure has been found in other instances

of transmission of resistant HIV-1These observations support the theory that

new infections involve transmission of a single predominant viral strain, rather

than the entire diverse quasi species generally present in chronically

infected individuals.

The reversion of particular mutations appeared to be associated with an

increase in viral RC, which implies that virus evolution to improve replication

occurred in the absence of antiretroviral drugs. In vitro competition

experiments

suggest that T215Y and M184V mutations in RT diminish replication competence

relative to the wild type in the absence of antiretroviral drugs. Similar

studies indicate that particular PI mutations, such as V82T, D30N, and L90M,

result in decreased viral replication capacity. In this study, viral fitness was

assessed using a recombinant assay of RC, which correlates well with other in

vivo and in vitro measures of viral fitness. In our patient, an increase in the

viral RC was associated with the reversion of T215Y to T215C in RT and with

the emergence of mixtures at 3 PR sites previously present as pure mutants

(L10L/F/V, I54I/V, and V82V/A). This finding suggests that mutations at 1 or

more

of these particular codons may decrease viral RC.

The correlation between genotypic and phenotypic resistance in this patient

also illustrates the fact that the presence of particular mutations that confer

resistance to one antiretroviral agent may result in increased sensitivity to

another drug. For example, a mutation at codon 184 (M184V) that is associated

with decreased susceptibility to lamivudine has been found in vitro to

sensitize virus to inhibition by zidovudine. In our patient, as resistance to

lamivudine decreased, resistance to zidovudine increased, which demonstrates

reciprocal interaction between susceptibility to these 2 drugs in a clinical

specimen. A similar sensitization to tenofovir by the 184 mutation has also been

suggested by in vitro studies and was seen in our patient's viral samples.

In this patient, infection with an HIV-1 isolate with multiple drug

resistance mutations led to rapid progression to AIDS. Despite multiple

mutations and

impaired RC, the virus load set point was high (>100,000 copies/mL). The

patient experienced a rapid decrease in CD4 cell count (temporally associated

with

reversion of drug resistance mutations) and an increase in in vitro viral RC.

The decrease in CD4 cell count and concomitant increase in RC were associated

with only a minor increase in plasma virus load, which suggests that the

immunologic deterioration was the result of an intrinsic change in the

pathogenicity

of the virus, rather than of a change in the virus burden. Additional

explanations for this individual's rapid progression to AIDS may include host

immunologic factors and changes in viral coreceptor use.

In summary, longitudinal analysis of viral evolution after transmission of

drug-resistant virus demonstrated stepwise back-mutation at specific sites,

rather than outgrowth of wild-type virus. Reversion of specific mutations in

viral

RT and PR was associated with an increase in HIV-1 RC. The rapid clinical

progression seen in this patient demonstrates that, despite initially impaired

viral fitness, an HIV-1 isolate with multiple drug-resistance mutations can

evolve to develop increased RC and significant pathogenicity.

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