WO1989006705A1

WO1989006705A1 - Plaque transfer assay for detecting retrovirus and measuring neutralizing antibodies

Info

Publication number: WO1989006705A1
Application number: PCT/US1989/000232
Authority: WO
Inventors: Ira Berkower; Dano Murphy
Original assignee: The United States Of America, As Represented By Th
Priority date: 1988-01-20
Filing date: 1989-01-23
Publication date: 1989-07-27
Also published as: JPH04505697A; EP0398986A1; EP0398986A4

Abstract

A plaque transfer assay for detecting retroviruses and measuring neutralizing antibodies, particularly against HIV-1 are described.

Description

PLAQUE TRANSFER ASSAY FOR DETECTING RETROVIRUS AND MEASURING NEUTRALIZING ANTIBODIES

BACKGROUND OF THE INVENTION

In the development of a safe and effective vaccine against the AIDS virus (HIV-1), an obstacle has been the difficulty of detecting viral inactivation by antibodies. This process is called "virus neutralization" and those antibodies which inactivate a virus simply by binding to it are called "neutralizing antibodies." An object of the present invention is. to easily and reliably detect neutralizing antibodies wherever and whenever they are found. For example, when a rabbit is immunized with a trial vaccine, the animal responds by producing antibodies which bind to the virus. But this does not necessarily indicate that these antibodies can neutralize the virus. The present invention for the first time provides an easy and reliable method of determining whether a given vaccine induces neutralizing antibodies. Similarly, a patient infected by HIV-1 may produce antibodies which could be detected by currently available tests. But again, whether these antibodies neutralize the virus and whether the level of the neutralizing antibodies changes at different stages of the illness can not be easily determined by the presently available tests.

SUMMARY OF INVENTION

It is, therefore, an object of the present invention to provide an assay for measuring the number of infectious viruses in a sample of retrovirus such as HIV (human immunodeficiency virus). It is a further object of the present invention to provide a highly sensitive assay for detecting and quantitating neutralizing antibodies present in a sample, such as an antisera. It is yet another object of the present invention to provide a method for determining the quality and level of neutralizing antibodies in a patient's sera at different stages of an illness and under a variety of clinical settings. A still further object of the present invention is to provide an assay for detecting limited viral propagation in the host cell monolayer. Other objects and advantages of the present invention will become evident from the following detailed descriptions of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein: Fig. 1 shows the results of plaque transfer assay of cell free virus; Fig. 2 shows HIV-1 propagation on T4 Ps₅ monolayer; Fig. 3 shows the linear relationship between the number of viruses present and plaque forming units(PFU); Fig. 4 demonstrates neutralization of cell associated virus; Fig. 5 demonstrates the neutralization of cell free virus; Fig. 6 shows the percent of HIV-1 neutralization at 1:200 dilution of each patient's serum; and Fig. 7 shows the cross reacting neutralization of cell-associated virus of different HIV isolates despite major variations in primary sequence.

DETAILED DESCRIPTION OF INVENTION

The above and various other objects and advantages of the present invention are achieved by a plaque transfer assay, comprising the sequential steps of: (a) culturing a monolayer of host cells infectable by virus, the number of which is desired to be determined; (b) infecting said monolayer of host cells with said virus and allowing the virus to be replicated by infected host cells; (c) transferring the monolayer to a nitrocellulose membrane; (d) treating said nitrocellulose membrane to release viral RNA; and (d) detecting the released viral RNA by a virus specific radiolabeled probe, the number of radiolabeled spots thus detected being a measure of the number of infectious virus. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. In the assay of the present invention, limiting numbers of viruses are used to infect a monolayer or "lawn" of host cells growing on the bottom of a flat culture dish. Each infected cell releases enough virus to infect the neighboring cells, until the original infected cell is surrounded by a large (1 to 2 mm) zone of infected cells. For other types of viruses which kill the infected host cells, this produces a hole in the monolayer. Each such hole is counted as a "plaque". But for certain viruses, such as HIV-1, which do not kill the monolayer cells, each infected zone must be detected by some other method, such as by a radioactive probe. Then each radioactively labeled zone or spot is counted as a "plaque". The number of plaques is generally proportional to the number of live viruses added to the culture. Thus, neutralizing antibodies which reduce the number of live viruses, can be detected by counting the reduction in the number of visible plaques compared to the number of plaques formed in the absence of antibodies. The method is very sensitive, because low number of viruses are added to each assay and because each plaque greatly amplifies the amount of viral material by the exponential growth of virus on the monolayer. Hence, even a few molecular events of virus neutralization results in the loss of a comparable number of microscopic plaques which can be easily counted. Heretofore, there were at least two major factors preventing the development of a plaque forming assay for 1 HIV-1. First, a suitable host cell which could support

2. the growth of the virus and still form a monolayer was

3 not available. This problem was solved by the

4 construction of a hybrid cell line by recombinant DNA

5 technology whereby the cloned gene for the T4 surface

6 receptor of HIV-1 was inserted into an adherant HeLa cell

7 (Madden et al, Cell, 47:333-348 1986). The resulting 8 hybrid cell line was found suitable for the purpose of 9 growing HIV-1, but a plaque forming assay for counting 10 live viruses and for measuring the neutralizing 11 antibodies remained yet to be developed. 12 The second problem related to the HIV-1 virus 13 which produced little or no visible damage in the host 14 cell. Hence, a new indicator of each zone of infection 15 was needed. Furthermore, such an indicator had to be 16 sensitive and highly specific in order to detect infected 17 cells in the presence of a background of uninfected 18 cells. In accordance with the present invention, this 19 problem is solved by using a sensitive method of DNA-RNA 20 hybridization with a P32 labeled virus-specific DNA probe 21 to detect viral RNA inside each infected cell of the22 monolayer. Thus, the assay comprises growing the virus23 on a monolayer of the hybrid cells, followed by transfer24 of the entire monolayer to nitrocellulose. Thereafter,25 the cells are lysed while still on the nitrocellulose 26 membrane releasing the viral RNA wherever there exists a27 zone of infection. The released RNA binds to the28 membrane and each zone of viral RNA on the membrane is29 detected by hybridizing the membrane with a P32 labeled30 DNA probe specific for the virus. Unbound P32 is washed31 off, and the membrane is exposed to photographic film,32 producing an exposed zone on the film corresponding to33 each zone of infection on the monolayer. The resulting 34 plaques are large enough to be seen even across the room. The specific steps of the method are now listed.

A. Growing host cells.

B . Treating the virus with antibodies.

C. Infecting a monolayer of host cells with virus.

D . Culturing the monolayer.

E. Harvesting the monolayer by transfer to nitrocellulose.

F. Processing the nitrocellulose membranes.

G. Labeling the virus-specific probe.

H. Hybridizing with the probe to detect viral plaques.

The details of the assay are now described.

A. Growth of cells

1. The T4Ps5 cell line is derived from an adherant HeLa cell line. It grows well in DMEM medium which consists of Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum (Reheis), 100 U/ml penicillin, 100 microgram/ml streptomycin and 10 microgram/ml geneticin (Gibco). The cells are split 1:10 every 4 to 5 days, or when they reach confluence. -For use in the assay, the confluent cells in one 75 cm flask are harvested, washed in the growth medium and resuspended in 5 ml of the medium. Twenty microliters are used for each culture, which is eventually cultured in a 6 cm culture dish. This amounts to a 1:80 dilution of the cells, which then require 10 to 14 days to reach confluence in the dish. This sequence allows enough time for the virus to replicate in culture, each assay being harvested when the cells reach confluence. 2. To illustrate, infected H9 cells were used which chronically carry and express the IIIB or RFII isolates of HIV-1 (Obtained from N.I.H., Bethesda, MD). The H9 cells are grown in RPMI 1640 supplemented with 15% fetal calf serum (Reheis) and 100 U/ml penicillin, 100 microgram/ml streptomycin, and 2 mM glutamine. The cells are fed every 4 days. When used for the assay, the cells are harvested, washed and resuspended in culture medium at 1 ×110⁶ cells/ml. They are irradiated with 10,000 Rad from a gamma source to prevent further proliferation.

B. Treating the virus with antibodies

1. Antisera or purified IgG fractions are diluted 1:4 in phosphate buffered saline, sterile filtered, and then serially diluted in 3-fold or 10-fold steps as required.

2. Typically about 40,000 chronically infected H9 cells are used as the source of virus, since this generally gives about 100 plaque forming units (PFU) per culture dish, which is both convenient to count and gives statistically significant results. Alternatively, cell-free virus from the culture supernatants of acutely infected H9 cells are used. The supernatants are culture titered prior to use, and the amount which gives 100 PFU is added to culture dish, generally about 40 to 100 microliters.

3. The virus (in 40 microliters) is added to 10 microliters of each dilution of antibody and incubated in the presence of the antibodies for 2 hours at 37°. The antibody treated virus is then added to the monolayer cells. If the antibody neutralizes the virus then little or no infectious virus remains after this step. Neutralization of virus is measured in terms of reduction in the number of plaques.

4. The titer of antibody is calculated based on the final dilution in the virus treatment step. For example, 10 microliters of a 1:4 dilution of serum plus 40 microliters of infected H9 cells gives 50 microliters of a 1:20 dilution of antibody. If half of the virus is killed at this dilution then the titer is 1:20.

C. Infecting the monolayer cells with virus.

1. The T4Ps5 monolayer is prepared for receiving the virus by activation with recombinant epidermal growth factor (Collaborative Research). The cells are pretreated with polybrene 2 μg/ml for 30 minutes at 22°C, then washed and incubated with recombinant EGF 100 ng/ml for 1 hour prior to the infection. It was found that this pretreatment increases the expression of the T4 receptor for the virus, and results in increased number of plaques at the end of culture. This host cell activation step is a unique feature of the present method and, at least partially, it contributes to the sensitivity of the assay.

2. The activated T4ps5 monolayer cells are mixed with the infected H9 cells or cell-free virus (after antibody treatment or untreated) and incubated for about 5 hours or overnight (about 14-16 hours) at 37°C in a 5% CO₂ incubator in a volume of about 0.25 ml. The incubation is in 5 ml polypropylene tubes. At the end of this infection period, 2.5 ml additional DMEM with 10% fetal calf serum is added to each tube and the contents are resuspended and transferred to a 6 cm diameter culture dish. D. Culturing the monolayer

The cells are cultured for 10 to 14 days in a 5% CO₂ atmosphere at 37°C, until the monolayer cells reach confluence. At the end of the culture, the culture supernatant is removed and is occasionally used to confirm production of viral antigens by the standard "antigen capture" radioimmunoassay.

E. Harvesting the monolayer by transfer to nitrocellulose

At the end of culture, the cell monolayer is washed with phosphate buffered saline, pH 7.4, and while still barely moist, a close fitting nitrocellulose membrane is gently pressed onto the surface of the dish to allow adherance of cells to the membrane. After 5 minutes, the membrane is gently peeled off the culture dish whereby the cells are lifted up with the membrane. Thus, complete transfer of the monolayer occurs including the infected cells with the virus they contain. Care must be taken at each step to avoid smearing the monolayer cells, so that the number, size and location of each infected center is fully preserved on the membrane.

F. Processing the nitrocellulose membrane

1. The membrane is turned over, so that the cell-side now faces up. It is then placed on a moist sheet of blotting paper which is saturated with 6% formaldehyde in 50% formamide in a 6 fold concentrate of saline with sodium citrate (6 X SSC). This lyses the cells on the nitrocellulose membrane without distorting the geometry of the zones of infected cells or damaging the viral RNA present in these cells. After 10 minutes at 65°C, the membrane is placed on a moist sheet of blotting paper saturated with 6 X SSC with 0.1% sodium dodecyl sulfate (SDS) for another 10 minutes at room temperature (about 22°-25°C). This reduces the level of cell debris on the surface of the membrane. The SDS is removed by a final 10 minutes incubation at room temperature on a third blotter paper saturated with 6 X SSC.

2. The treated nitrocellulose membrane is air dried and then baked for 2 hours in a vacuum oven at 80°C. This permanently fixes the RNA to the membrane. The pattern of viral infection on the monolayer, including the number of infected centers and their size and distribution, is represented on the nitrocellulose membranes by discrete zones of viral RNA which is then detected by standard hybridization technique using a virus-specific probe.

G. Labeling the virus specific probe.

1. The virus specific probe comprises a nearly full length fragment of the IIIB isolate of HIV-1. It extends from a Sst I site to a Bam HI site, is 9.4 Kb in length and includes sequences for the gag, pol, and env genes of the virus (obtained from the Center for Biologies, FDA). It is grown in E. coli as an insert in the plasmid pCGlO-Ecogpt. The plasmid DNA is isolated, cut with the restriction enzyme BamHl, and the probe purified as a 9.2 Kb band on a agarose gel.

2. The probe is P32 labeled by the standard nick translation method using the kit purchased from New England Nuclear. However, any other labeling technique well known to one of ordinary skill in the art can also be employed, such as biotin labeled nucleotides, synthetic oligonucleotides and the like. The reaction mixture contains 10 microliter of alpha-P32 dCTP, 5 microliter of nick translation buffer (Tris-HCl pH 7.4, MgCl2, BSA, dithiothreitol), 5 microliter of cold dATP, dTTP, and dGTP (all 100, μM), 5 microgram of DNA probe, 2 microliter of DNA polymerase I (1.2 units), and 2 microliter of pancreatic DNase I (0.4 units). The mixture is incubated for about 2 hours at about 14°C and then passed over a 4 ml Sephadex G-50 column to separate the products from the reagents. Finally, the P32 labeled probe is added to the hybridization buffer (vide infra) and boiled for about 5 minutes to generate single stranded DNA for hybridization.

H. Hybridizing with the probe to detect viral plaques

1. The baked membranes are prehybridized with unlabeled herring sperm DNA to reduce the background of nonspecific hybridization. They are washed in 6X SSC for 30 minutes at 65°C and then prehybridized overnight in: 6 X SSC, 10% dextran sulfate, 5% Denhart's solution, 0.2% SDS, 1 microgram/ml poly A, and 300 microgram/ml herring sperm DNA. The prehybridizing mixture is kept in a sealed plastic pouch in a 65°C waterbath.

2. The membranes are then incubated overnight in the same buffer used in prehybr.idization, except that the herring sperm DNA concentration is reduced to 200 microgram/ml and the single stranded P32 labeled probe is added. During this step, the P32 labeled probe finds and binds to viral RNA immobilized on the membrane. Each zone of infection in the original monolayer corresponds to a zone of viral RNA on the nitrocellulose membrane which becomes P32 labeled with the radioactive labeled probe in this step.

3. Any probe which remains on the membrane due to nonspecific binding must be washed off to reduce the background. This is accomplished by washing the membranes extensively as follows: First, the membranes are washed twice with 2 X SSC containing 1% of SDS at room temperature (22°-25°C) for about 5 minutes each time, twice in 2 X SSC containing 1% SDS for 30 minutes at 55°C. Then they are washed in 0.2 X SSC with 0.1% SDS for 30 minutes at 55°C, followed by another wash in the same buffer for 2 minutes at room temperature. Then they are air dried. These washes reduce the background to the point that the uninfected control monolayer gives essentially no signal.

4. Finally, the zones of viral RNA which bind the P32 labeled probe are detected by exposing the P32 labeled nitrocellulose membranes to suitable photographic film for 1 to 3 days in an X-ray cassette with image intensifier screens at -70°C. When the film is developed, the plaques are read as black dots on the film (Figure 1). The remarkable feature of this assay is that the plaques are discrete, macroscopic and unequivocal. This makes them easy to count by unaided eye and the assay consistently gives a reproducible number of plaques for a given input of virus.

The assay of the present invention is useful in various ways some of which are now listed: A. Detection and quantitation of live retroviruses.

B . Detection and quantitation of neutralizing antibodies.

C. Systematic search for and development of a vaccine against HIV-1

D . Evaluation of the quality of antibodies induced by any vaccine candidate, and prediction of live virus challenge studies in primates.

E . As a clinical laboratory test for determining neutralizing antibodies in infected patients at different stages of illness.

F . As a general virus neutralizing assay which can detect limited viral propagation in the host cell monolayer.

These utilities are now exemplified.

EXAMPLE - 1

Virus detection and quantitation The assay is highly effective in detecting small numbers of human immunodeficiency viruses. When the culture supernatants of acutely infected cells are used, it consistently yields discrete macroscopic plaques. Unlike any other plaquing assay for HIV, it uses the whole virus, not just one component or one function of the virus. Hence it allows calculation of the plaque forming titer directly from the number of plaques at a given dilution of the culture supernatant. A typical result is shown in Fig. 1. Decreasing amount of the infectious culture fluid are added to the monolayer cells as described herein above and the plaques are developed as shown. The low background is noteworthy when no virus is added. Also the large size of the discrete plaques in this life sized print should be noted. For the first time the assay of the present invention makes it possible to count HIV plaques with the unaided eye. The number of plaques can be counted in a reproducible and reliable manner, giving a titer of about 1.5 × 10³ PFU/ml in the example shown. Plaque formation depends on the growth of the virus on the monolayer. As shown in Fig 2, viral antigens (as measured by radioimmunoassay) are released into the culture supernatant over the course of the culture and the greater the input of virus, the more antigen is produced, and the greater number of plaques formed. However, even a very high dose of virus fails to produce any antigen and no plaques are formed when the virus is used to infect a control monolayer such as CV-1 which lacks the T4 receptor for virus and cannot support its growth. Thus, a cell culture supporting the viral growth is required. Under suitable conditions, culture supernatant as measured by radioimmunoassay, correlates linearly with the number of plaques which increases linearly with the amount of virus added (Fig 3). This is important because it indicates "single hit kinetics" ie_, that each plaque is the result of infection by a single virus. If two viruses were required, the concentration dependence would increase with the second power of the virus concentration. This indicates that the plaques are highly sensitive to neutralizing antibodies, since the neutralization of a few viruses would result in the loss of a similar number of the macroscopic plaques. Thus, the unique assay of the present invention gives a highly reproducible digital readout of molecular events expanded to the level of visible plaques. The versatility of the new assay is shown by the fact that cell-free virus (Figs 1 and 3) or cell- associated virus (Fig 2) can be used. It is noted that the assay of the present invention is the only HIV plaquing assay which can measure cell associated virus. The use of cell-associated virus is convenient because the amount of virus added to the culture can be standardized based on the number of infected cells added per assay. Typically 40,000 infected H9 cells are used to give about 100 plagues. An additional advantage of the cell-associated virus is the lack of nonspecific killing with normal rabbit serum which is characteristically observed with cell-free virus. This permits testing of candidate vaccines which would be difficult to measure with cell-free virus.

EXAMPLE - 2

Detection and quantitation of neutralizing antibodies Using the new assay, remarkable success is achieved in detecting human neutralizing antibodies against HIV. Plaque formation from cell-associated or cell-free virus is readily inhibited by serum from most seropositive patients. For instance, Fig. 4 shows the results when the cell-associated virus is incubated with normal control sera at a dilution of 1:20 or 1:200 or with comparable dilutions of sera from three patients who were seropositive for antibodies to HIV-1. The normals (2,3, and 7) had no effect on the virus, while the seropositives (4,8, and 9) gave nearly total inactivation of virus at both dilutions. In fact, patient 9 gave a titer of 1:6000, as defined by 50% inactivation of virus. Similarly, patients 4 and 9 gave total inactivation of cell-free virus (Fig 5) while normal donor 2 had no effect. In further studies, plaque formation by cell-associated virus was uninhibited by 10 normal sera or by 5 sera from patients who were infected with the closely related retrovirus HTLV-I, which demonstrates the specificity of neutralization (Fig 6). Nineteen sera from seropositives (including the three in Fig 4) showed complete viral inhibition. Sera from 5 patients with clinical AIDS neutralized cell-associated virus only slightly less well than did sera from 14 asymptomatic seropositives. Neutralizing titers ranged from 1:60 to 1:6000 in the seropositive group, with a median titer of about 1:200. Thus, human neutralizing antibodies are quite prevalent among seropositive patients and can prevent transmission by cell-associated as well as cell-free virus. This assay is the only one capable of detecting neutralization of cell-associated virus, which is the presumed means of transmission in many cases. The results presented herein are quite significant in the search for an effective vaccine, since it demonstrates that humans are capable of producing neutralizing antibodies when immunized appropriately. A critical question in the AIDS vaccine field is whether the vaccine must contain numerous variants of the virus proteins in order to be effective against each of the many pathologic variants of the AIDS virus isolated so far. For example, the most divergent isolates identified so far are the IIIB isolate from New York and the RFII isolate from Haiti. The virus envelope proteins differ in these variants and are likely to require a different set of neutralizing antibodies. However, as shown in Fig 7, the same antiserum neutralized both virus isolates at the same titer. The cross reaction of neutralizing antibodies between these two highly divergent HIV-1 isolates denmonstrates for the first time that a vaccine containing only a few serotypes may be sufficient to prevent infection by multiple isolates. The flexibility of the present assay is of remarkable significance in testing neutralizing antibodies against new virus isolates as they become available and in identifying additional virus serotypes.

EXAMPLE - 3

The major commercial application of this assay may be in the development of a vaccine, particularly in identifying viral components which carry neutralizing determinants, testing immunogencity in animals and man, and in challenging the immune animal with live virus. The HIV vaccine is likely to be a recombinantly expressed viral protein which contains sites recognized by neutralizing antibodies. The approach to indentifying this protein in accordance with the present invention is to absorb the human neutralizing antibodies with each individual viral component as expressed by recombinant DNA methodology. If a given viral protein contains the neutralizing site, it will absorb out the neutralizing antibodies. When the absorbed antibody is added to the present assay, it will no longer be able to neutralize the virus, so the number of plaques will return to the untreated value. Because the present assay is the only plaque forming assay employing intact whole virus, it is the only one capable of detecting potential neutralizing antibodies to each one of the viral components. Other assays such as the VSV pseudotype assay can only measure neutralization by antibodies directed to the envelope protein, since the envelope protein is the only HIV protein incorporated into the pseudotype virus used in the assay. The second step in vaccine development is to test the vaccine antigen for immunogenicity, the ability to induce specific antibodies. The present assay is most useful for determining not only immunogenicity, but also for evaluating the neutralization quality of antibodies induced by the candidate vaccine as illustrated herein supra.

Evaluation of antibody quality and guiding the immunization of chimps for protection against live virus challenge. As demonstrated herein above, the present assay can determine whether the antibodies elicited by any vaccine are neutralizing antibodies. If more than one viral component is selected for development of a vaccine in the example above, then it may be possible in this step to pick which one of these candidates is the most immunogenic. If more than one is immunogenic, then the assay of the present invention can be employed to pick the best antigen, such as the least variable component among the different HIV-1 isolates, so that fewer serotypes would need to be included in the vaccine. Using immunized rabbits, we have shown that a vaccine consisting of just the viral envelope protein can elicit neutralizing antibodies. But the envelope protein is the most polymorphic viral component, so other viral components which are less polymorphic can now be tested for neutralizing determinants and immunogenicity. The third step in vaccine development is to immunize a susceptible host with a vaccine candidate and then challenge the host with live virus. The assay of the present invention is useful in this step by determining whether the host has made neutralizing antibodies and is ready for challenge. Clearly, a host with neutralizing antibodies should have a better chance of protection against infection by the live virus. To date, no candidate vaccine has passed this trial. EXAMPLE - 5

A clinical laboratory test for neutralizing antibodies. The relationship of the level of neutralizing antibodies as a definitive predictor of progression from asymptomatic seropositive to clinical AIDS or when they will develop the disease is not yet known. Groups of seropositive patients, such as hemophiliacs, who are known to have a long latency of infection may also have high levels of neutralizing antibodies; other groups such as infected children who are known to have a short latency may have lower levels of neutralizing antibodies. If so, the assay of the present invention allows to establish the biologic relevance of neutralizing antibodies and the onset of clinical AIDS. It should be noted that progression of the disease is very different from protecting against the infection in the first place. A vaccine would most likely work before exposure to prevent infection, but might be ineffectual against the illness after infection, as is generally the case. Another clinical use of this test is to resolve certain cases of seropositive individuals with an "indeterminate" western blot. In such cases, a positive neutralizing assay would be strong evidence of infection with HIV-1.

EXAMPLE - 6

A general method for detecting neutralizing antibodies against viruses with limited proliferative capacity on monolayer cells. A number of pathogenic viruses have not yet yielded to be grown in culture, including, for example, 1 hepatitis B virus. The virus may be capable of infecting

2 and persisting in a suitable host cell, such as a

3 hepatoma cell line, but may not produce enough virus to

4 be detected by conventional radioimmunoassays. However,5 a sensitive plaque transfer assay such as demonstrated

6 herein, with suitable probes specific for hepatitis B

7 virus DNA, would be an ideal screening method to find the

8 right host cell line and conditions for analyzing short

9 term virus growth in culture. Once this is found,

10 neutralizing antibodies could be analyzed as shown

11 herein. This might explain the protective effect of

12 hepatitis B surface antigen as a vaccine and why certain

13 people develop the chronic carrier state or chronic

14 active hepatitis. In addition, the virus receptor on the

15 cell surface could be studied, as well as the molecular

16 biology of virus replication and gene expression. Other

17 viruses such as HIV-2, could be studied in a similar

18 manner as suitable specific probes become available.

19 It is understood that the examples and embodiments

20 described herein are for illustrative purposes only and

21 that various modifications or changes in light thereof

22 will be suggested to persons skilled in the art and are

23 to be included within the spirit and purview of this

24 application and scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. An assay for determining the number of infectious virus in a sample, comprising the sequential step of:

(A) culturing a monolayer of host cells infectable by virus, the number of which is desired to be determined;

(B) infecting said monolayer of host cells with said virus and allowing the virus to be replicated by infected host cells;

(C) transferring the monolayer to a nitrocellulose membrane;

(D) treating said nitrocellulose membrane to release viral RNA; and

(E) detecting the released viral RNA by a virus-specific radiolabeled probe, the number of radiolabeled spots thus detected being a measure of the number of infectious viruses.

2. The assay of claim 1 wherein said virus is HIV-1 or HIV-2.

3. The assay of claim 2 wherein said virus is HIV-1.

4. The assay of claim 1 wherein the number of virus is determined by unaided eye.

5. An assay for determining the level of neutralizing antibodies against virus, in a biological sample, comprising reacting an aliquot of said biological sample with virus against which said antibodies were produced, for sufficient time to allow binding of said antibodies with antigenic sites of said virus and then performing the assay of claim 1 with and without the antibody-treated virus, the decrease in number of infectious virus seen in the assay with antibody-treated virus compared to untreated control being a measure of the level of neutralizing antibodies present in the biological sample.

6. A method for diagnosing the clinical status of HIV infection in a patient infected with HIV, comprising determining the level of HIV-antibodies in said patient at different clinical stages by the method of claim 5.

7. A method for determining antigenic component for the preparation of a vaccine, comprising identifying a specific neutralizing epitope by absorbing a known neutralizing antibody preparation with a viral antigen which completely depletes the neutralizing antibodies, the antigen completely depleting the neutralizing antibodies being vaccine candidate of choice; then employing said antigen to induce neutralizing antibodies in a host responsive to said antigen and then determining the level of neutralizing antibodies by the method of claim 5.