Distinguishing Between HIV 1 and 2

Extracted from a debate in the British Medical Journal

If there were two unique retroviruses, "HIV-1" and "HIV-2", as the "HIV" experts claim, then it should be easy "to distinguish between HIV-1 and HIV-2 infection" by the antibody tests and definitely by PCR.  The fact that Christopher Noble could cite only one such reference suggests this is not the case.

 

In the Kannangai et al study2 cited by Noble, according to the Western blot (immunoblot) and PCR status the patients were divided into 5 groups (Table 1).  All were re-tested using an "HIV-2 specific env peptide" ELISA consisting of "11 amino acid sequences from the immunodominant epitope", synthesised by Kannangai et al.

 

Table 1. The mean, standard deviation of absorbance values for different categories of samples tested by HIV-2 specific

 synthetic peptide ELISA and its correlation with HIV-2 PCR a,b. 

Category of samples

No. of samples

ELISA absorbance

Peptide ELISA result

Percentage Concordance

 

 

Mean

SD

POS

NEG

 

HIV-1 PCR and IB POS

30

0.040

0.05

1

29

97

HIV-2 PCR and IB POS

19

1.669

0.735

19

0

100

HIV-1&2 and IB POS

5

1.119

0.769

5

0

100

HIV-2 IB POS; HIV-1&2 PCR NEG

2

0.387

0.388

1

1

50

HIV-1&2 IB POS; HIV-2 PCR NEG

7

0.015

0.0045

0

7

100

a SD, standard deviation; IB, immunoblot; POS, positive; NEG, negative.
b ELISA negatives (n=45) mean OD±SD: 0.056±0.040 (range 0.003–0.157)

 

There are several reasons why one cannot claim Kannangai et al have proven "the ability of both antibody and PCR tests to distinguish between HIV-1 and HIV-2 infection".

 

The first and absolutely necessary condition which Kannangai et al had to satisfy was to have patients which were proven to be infected with either "HIV-1", "HIV-2" or dually infected by means other than their test.  That is, the patients in each of the five groups were indeed infected with the claimed viruses.  However, they could not have obtained such proof.

1. According to Hans Gelderblom “…the bulk of internal proteins of the virion [retroviruses] with molecular weight (mw) between 10,000 d and 30,000 d are group-specific (gs) for viruses originating in a given animal species (gs-spec. antigens). The major protein constituent of mammalian C-type oncornaviruses [retroviruses] with a molecular weight in the range of 30,000 d was found to possess, besides gs spec. antigen, an antigenic determinant that is shared by C-type viruses of many mammalian species including monkeys and was thus termed gs interspecies (gs-interspec.) antigen".3  This leads to antibody cross-reactivity with gag proteins/antigens.  It is accepted that "HIV-1" antibodies cross-react with the HIV-2 antigens and vice versa, the "HIV-2" antibodies cross-react with the "HIV-1" antigens.4  According to Kannangai et al "evidence is emerging that many HIV-1 and HIV-2 dual infections detected by immunoblot may actually be HIV-1 infections with cross-reactivity for HIV-2 bands".

 

According to Gallo  “the antibodies which react with retroviral glycoproteins are not directed against the proteins “but against the carbohydrate moieties on the molecule that are introduced by the host cell as a post-transcriptional event, and which are therefore cell-specific and not virus-specific”.5  According to other eminent retrovirologists including Robin Weiss and Reinhard Kurth “we conclude that the majority (if not all) of normal human sera contain naturally occurring heterophile antibodies that react with the carbohydrate moieties of retrovirus envelope antigens”.6  In a study published in 1996 it was reported that up to 8% of "non-infected blood donors had reactivity to nonglycosylated gp41 epitopes". 7  Obviously since sick individuals have elevated levels of antibodies the prevalence of such reactivity would be much higher.

 

Given the above facts one would have to conclude that Kannangai et al had no proof:  (i) how many, if any, of their patients having a positive immunoblot were infected and with which virus, "HIV-1", "HIV-2", or both to begin with;  (ii) how many, if any, of the patients who had a positive "HIV-2" peptide ELISA were indeed infected with this virus.

 

2. Kannangai et al wrote:  "The HIV-1 and the HIV-2 specific nested PCRs were carried out as described previously (Delwart et al., 1995; Damond et al., 1998)”.  In Damond et al8 one reads "HIV-2 is rarely isolated from peripheral blood mononuclear cells (PBMCs) [from HIV-2 antibody positive individuals] and the detection rates of proviral DNA by PCR amplification vary between 50 and 80%…"  Because of the low correlation between the antibody and the PCR tests, Damond et al "evaluated a new human immunodeficiency virus type 2 (HIV-2) DNA amplification strategy based on peripheral blood mononuclear cell long PCR (XL PCR) followed by nested PCR amplification.  The primers used were located in the highly conserved long terminal repeat in the pol regions of the genome.  Five primer pairs corresponding to different regions of the HIV-2 env gene were used in the nested step". Samples from 42 HIV-2 positive patients were tested and amplification with one primer was considered a positive PCR.

 

If a positive HIV-2 antibody test was proof for HIV-2 infection and if the primers were amplifying the env gene, then:  (i) each primer pair should have given positive results with all 42 samples;   (ii) all samples should have tested positive with all primer  pairs.  This is not the case.  Two samples were not amplified by any of the five primer pairs. Two samples were amplified by only two primer pairs;  Four samples were positive with three primer pairs;  five samples were positive with four primer pairs and twenty nine with five primer pairs.

 

 Numbers of the 42 samples amplified by 2/5 - 5/5 primer pairs

No. of primer pairs

2

3

4

5

Samples

2

4

5

29

 (An additional 2 samples were NOT amplified by any of the 5 primer pairs)

 

Comment:  Since both the PCR and the antibody tests are very sensitive, if they detect HIV-2 a perfect correlation should exist between the PCR and the antibody test as Christopher Noble claims.  However:  (i) as mentioned, the PCR is positive only in 50-80% of antibody positive patients;  (ii) since Damond et al spared no effort to obtain a positive PCR and since they used a highly sensitive "strategy" which allowed them to bypass HIV-2 "diversity", if the amplified DNA was HIV-2, all patients should have had a positive result with all primers.  Instead, 26% (11/42) of the patients had a positive result with some but not all primers, and in 5% (2/42) the results were negative with all primers.  As mentioned, amplification with even one primer was considered a positive PCR.  However, the finding of a positive PCR with a few primers, or even with all five primers if they do not amplify the entire env gene, is not proof the full env gene is present much less the entire HIV genome, that is, HIV infection.  Since Kannangai et al used the same HIV-2 PCR method as Damond's et al then, given the many shortcomings of this method, one must conclude that neither group had proof that a positive PCR was proof for HIV-2 infection.

 

It must also be pointed out that the Kannangai et al study was not blind and the number of patients tested at least in some groups was extremely low.

 In conclusion, at present:  (i) there are no "antibody and PCR tests [which are able] to distinguish between HIV-1 and HIV-2 infection";  (ii) there is no proof for a correlation between the HIV antibody and PCR tests;  (iii) there is no proof that HIV-1 and HIV-2 infection can be defined in molecular terms.

 

2. Kannangai R, Ramalingam S, Prakash KJ, Abraham OC, George R, Castillo RC, et al. A peptide enzyme linked immunosorbent assay (ELISA) for the detection of human immunodeficiency virus type-2 (HIV-2) antibodies: an evaluation on polymerase chain reaction (PCR) confirmed samples. Journal of Clinical Virology 2001;22:41-6.

3. Bauer H, Daams JH, Watson KF, Molling K, Gelderblom H, Schafer W. Oncornavirus-like particles in HeLa cells. II. Immunological characterization of the virus. Internat J Cancer 1974;13:254-261.

4. Gnann JW, Jr., McCormick JB, Mitchell S, Nelson JA, Oldstone MB. Synthetic peptide immunoassay distinguishes HIV type 1 and HIV type 2 infections. Science 1987;237:1346-9.

5. Kalyanaraman VS, Sarngadharan MG, Bunn PA, Minna JD, Gallo RC. Antibodies in human sera reactive against an internal structural protein of human T-cell lymphoma virus. Nature 1981;294:271-273.

6. Lower J, Davidson EA, Teich NM, Weiss RA, Joseph AP, Kurth R. Heterophil human antibodies recognize oncovirus envelope antigens: epidemiological parameters and immunological specificity of the reaction. Virol 1981;109:409-17.

7. Sayre KR, Dodd RY, Tegtmeier G, Layug L, Alexander SS, Busch MP. False-positive human immunodeficiency virus type 1 western blot tests in noninfected blood donors. Transfusion 1996;36:45-52.

8. Damond F, Loussert-Ajaka I, Apetrei C, Descamps D, Souquiere S, Lepretre A, et al. Highly sensitive method for amplification of human immunodeficiency virus type 2 DNA. J Clin Microbiol 1998;36:809-11.