Treatment of SARS with human interferons

[Guest guest]

Effective antiviral agents are needed to treat severe acute

respiratory syndrome-associated coronavirus (SARS-CoV) infection. We

assessed the antiviral potential of recombinant interferons against

two clinical isolates of SARS-CoV--FFM-1, from Frankfurt patients,

and Hong Kong--replicated in Vero and Caco2 cells. Interferon ß was

five to ten times more effective in Caco2 cells. Interferon

effectively inhibited SARS-CoV replication, but with a selectivity

index 50-90 times lower than that for interferon ß. Interferon was

slightly better than interferon in Vero cell cultures, but was

completely ineffective in Caco2 cell cultures. Interferon ß could be

useful alone or in combination with other antiviral drugs for the

treatment of SARS.

 

Lancet 2003; 362: 293-94

 

Several clinical isolates of the severe acute respiratory syndrome-

associated coronavirus (SARS-CoV) have been isolated, including FFM-

1 and FFM-2 from SARS patients admitted to the Clinical Centre of

Frankfurt University.1 So far, no specific treatment has been

identified for SARS, but attempts are being made to identify

antiviral agents that could be effective in treating the SARS-CoV

infection.2

 

We assessed the antiviral potential of recombinant interferons , ß,

and against two clinical isolates (FFM-1 and Hong Kong: obtained

from Dr Niedrig, Robert Koch Institute, Berlin) of SARS-CoV. We used

commercially available interferons (Intron A [interferon -2b], Essex

Pharma, Munich, Germany; Betaferon [interferon ß-1b], Schering,

Berlin; and Imukin [interferon -1b], Boehringer, Ingelheim) that

have been used for their antiviral, antitumour, and immunomodulating

activities. We visually scored cytopathogenic effects induced by the

virus 72 h after infection in 96-well microplates on confluent

layers of Vero and Caco2 cells, the latter derived from human

colonic tumour, treated with interferons 24 h before and immediately

after virus infection (mutiplicity of infection 0·01). The

selectivity index was defined as the ratio of the concentration of

the interferon that reduced cell viability to 50% (CC50) to the

concentration of the compound needed to inhibit the cytopathic

effect to 50% of control value (EC50). We assessed the cytotoxic

effects of the drugs with an MTT cell-proliferative Kit I (Roche,

Mannheim, Germany). Viral titres in supernatants collected from

infected cultures 72 h after infection were measured by the 50%

tissue culture infective dose (TCID50) calculated from

cytopathogenic effects induced in monolayers incubated with varying

dilutions of the supernatants. To validate the interferon effects on

SARS-CoV, we included vesicular stomatitis virus (strain Indiana),

replication of which is extremely sensitive to interferon action.

 

Interferon ß was the most potent inhibitor of SARS-CoV (table). In

Vero cells, selectivity index of interferon ß activity for SARS-CoV

strain FFM-1 was 50-fold and 25-fold higher than that of interferon

and interferon , respectively. Sensitivity of the Hong Kong isolate

was similar to that for the FFM-1 strain. Interferon ß (EC50 105

IU/mL) was more potent than interferons or (EC50s 6500 IU/mL and

1700 IU/mL, respectively). Vesicular stomatitis virus strain Indiana

was much more sensitive to interferon treatment than SARS-CoV;

interferon ß had the highest antiviral activity in Vero cell

cultures (EC50 18 IU/mL).

 

---

Mean (SD) EC50 (IU/mL)* CC50 (IU/mL) Selectivity index

SARS-CoV FFM-1 SARS-CoV Hong Kong VSV Indiana SARS-CoV FFM-1 SARS-

CoV Hong Kong VSV Indiana

Vero†

Interferon 4950 (890) 6500 (980) 638 (98) >10000 >2 >1·5 >16

Interferon ß 95 (17) 105 (21) 18·0 (3·7) >10000 >105 >95 >556

Interferon 2500 (340) 1700 (290) 508 (62) >10000 >4 >5·9 >20

Caco2†

Interferon 1530 (220) 880 (130) 3700 (420) >10000 >6·5 >11·4 >2·7

Interferon ß 21·0 (3·9) 9·2 (2·1) 1030 (150) >10000 >476 >1087 >9·7

Interferon >10000 >10000 >10000 >10000 NC NC NC

VSV=vesicular stomatitis virus. NC=not calculable *Mean (SD) of

eight assays. †Cells treated with interferons 24 h before and during

virus infection.

Effect of Interferons on virus-induced cytopathogenic effects of

SARS-CoV and vesicular stomatitis virus

 

---

 

We studied the action of interferons on SARS-CoV-induced

cytopathogenic effects in Vero cells when the drugs were added after

infection (1 h adsorption period). Interferon ß was active against

FFM-1 strain (EC50 560 IU/mL) but interferons and were not.

Therefore, only interferon ß can be used as an antiviral agent after

infection, even though its EC50 value shows a six-fold increase over

the value obtained in pretreated cultures (table).

 

We assessed also the relevance of inhibition of virus replication

for suppression of virus-induced cytopathogenic effects in cultures

treated with interferon ß 24 h before and immediately after virus

infection. We infected Vero cells with FFM-1 strain at multipicity

of infection 0·01 and assessed viral replication by peroxidase

staining with immune serum after treatment with different

concentrations of interferon (figure). Native cells, or cells fixed

with 60 parts methanol to 40 parts acetone were assessed 72 h after

infections. We also quantified the genomic titre (RNA copies/mL)

with real-time PCR. Interferon ß showed a dose-dependent inhibition

of the production of infectious virus in culture. In the absence of

virus-induced cytopathogenic effects, however, interferon ß

treatment did not completely reduce virus titre and antigen

production. In cultures treated with 10 000 IU/mL, a reduction of

about 5x104-fold was noted compared with the untreated control

(1·9x102 vs 9·7x106 TCID50/mL). The number of gene copies are

reduced by interferon ß in a dose-dependent way; however, the

magnitude is around 3·3-4·5 log higher than the infectious virus

titre measured in cell culture. This difference may be due to the

presence of non-infectious viral RNA or the overexpression of the

sequence amplified in our real-time PCR system. Similar observations

have been made by Garcia and colleagues3 on the quantitative

detection of Rift valley fever virus gene copies to assess the

antiviral effect of drugs.

 

Effect of interferon ß on replication of SARS-CoV strain: FFM-1 in

Vero cells 72 h after infection

 

Virus detected in serum with peroxidase staining. *Values represent

mean (SD) from three experiments, each done in triplicate.

 

Interferons and ß share components of the same receptor and are

type I interferons. Interferon uses a different receptor system and

is a type II interferon. We noted that interferon ß was by far more

effective than interferon against SARS-CoV and vesicular stomatitis

virus. Although interferons and ß primarily induce the same

proteins, some proteins may be induced by one better than the other.

By use of oligonucleotide arrays, Der and colleagues4 identified the

genes differentially regulated by interferons. They reported more

than a 21-fold increase of MxA protein by interferon , more than a

31-fold increase by interferon ß, and none by interferon . Gene-

array analysis of Caco2 cells infected with FFM-1 in our laboratory

has shown a nine-fold increase of MxA gene expression in cultures 24

h after infection. This increase could be one of the reasons for

amplification of interferon ß effect that yields the higher

antiviral potential.

 

Interferons and , alone or in combination, are partly effective

against animal coronaviruses, mouse hepatitis virus, and

transmissible gastroenteritis virus; a few data on human studies

report a limited response of interferon by the intranasal route.5

We showed that interferons inhibit SARS-CoV replication in vitro.

Interferon ß was most potent, showing prophylactic protection and

antiviral potential after infection. Interferon ß could be the drug

of choice, alone or in combination with other antiviral drugs,2 in

the treatment of SARS.

 

Direct link is:

http://www.thelancet.com/journal/vol362/iss9380/full/llan.362.9380.or

iginal_research.26622.1

 

Attilio