2. What is HIV ?
• HUMAN IMMUNODEFICIENCY
VIRUS
• Belongs to retroviruses.
• Causes AlDS.
• Acquired Immunodeficiency
Syndrome
History/Origin
First case - New York (USA) in 1981;
Isolation of HIV-I from Pasteur
instiute, Paris in 1983.
3.
4. RETROVIRUSES
Retrovirus – possess reverse transcriptase .
viral RNA - DNA
Two genera - Pathogenic to humans .
Genus Lentivirus: (HIV)-1 and (HIV)-2.
Genus Deltaretrovirus: (HTLV- 1)
Family - Retroviridae
Three subfamilies
Seven genera
5.
6. HIV in humans
Acquired from
chimpanzee (Pan
troglodytes
troglodytes)
By cross species
infections of SIV
cpz from rural
Africa .
From where HIV came ??
7. Morphology - Shape ???
Spherical. 80- 110 nm.
Envelope
Matrix layer
Nucleocapsid.
Lipid part: host cell membrane derived.
Protein part: two components:
1. Glycoprotein: 120 (gp 120):
projected as knob like spikes on surface .
2. Glycoprolein: 41 (gp 41):
They form anchoring transmembrane pedicles.
Envelope
8. Capsid :Icosahedral in symmetry,
Made up of core protein.
Inside, there is a dense cylindrical inner
core which encloses:
RNA: two single-stranded + sense
linear.
Viral enzymes
reverse transcriptase,
integrase
proteases
https://www.youtube.com/watch
?v=CUV_KUr7418
Matrix :
Protein layer beneath envelop .
Nucleocapsid:
9.
10. Rna has HIV Genes Antigens
• Three structural genes- gag, pol, and env.
• Six non-structural or regulatory genes.
Env gene - codes
envelope glycoprotein (gp 160
gp 120: main receptor - binds to
CD4.
gp 41: fusion protein.
Pol gene codes
reverse transcriptase, protease
and integrase p32(integrase),
p51(reverse transcriptase) and p66
.
Gag gene codes –
core and shell.
p 17 constitutes the matrix or shell
antigen .
p24 and p15- constitute the core
antigens .
Gene Proteins Antigens
11. Non-Structural Genes
• Tat - transcriptional trans activator gene,
for HIV-I replication.
• Nef(negative factor gene):
CD4 expression on the host cell surface.
• Rev (regulator of virus gene):
structural proteins.
• LTR (long terminal repeat) sequences –
provide promoter, enhancer and
integration signals.
• Vif (viral infectivity factor gene):
infectivity of viral particles.
• Vpu gene: It promotes the CD4
degradation and release of progeny
viruses from; expressed only by HIV-I.
• Vpr gene: transport of viral genome in
nucleus and arrests host growth.
• Vpx is found in HIV-2 (and SIV), but
not in HIV-1.
Non-structural genes - regulate viral replication.
12.
13. Antigenic Variation and Diversity
• Because of undergoing high rates of mutation.
• Due to the error prone nature of reverse transcriptase enzyme.
Mutations may occur in any genes, mostly in env gene.
• Envelope proteins is major target against which antibodies are produced.
Hence mutations in env gene is the main reason which explains
Why ????
HIV evades the host's immune response.
Vaccination against HIV is extremely difficult.
14. HIV Serotyping - Based on
Sequence differences in env gene.
Two serotypes HIV-1 and 2
Recently, a HIV strain related to
gorilla SIV was identified in a
Cameroonian woman in 2009 and
has been proposed as group P.
Circulating recombinant forms" or
CRFs derived from recombination
between different subtypes..
For example, CRF01_AE is a
recombination between subtypes A
and E
HIV-1
Three distinct groups (M, N, and O).
'M' is the dominant group worldwide - ten subtypes or clades (A-J).
Further split into sub-subtypes such as A1 and A2 or F1 and F2.
HIV-1 subtypes -differ in geographical distribution and
transmission.
16. Subtype A
✓ common in West Africa.
Subtype B
✓ Europe, America, Japan, and
Australia.
Subtype C
✓ common form worldwide (47%)
Africa, India, and China.
In Cameroon (West Africa), all known HIV
groups and subtypes are found. It is
probably, considered as the place of origin of
the virus.
Asian and African subtypes (C and
E)
✓ transmitted heterosexually
American strains (subtype B)
✓ Preferentially spread through
blood
✓ Homosexual contact.
Geographical distribution Transmission:
17. HIV-2
• Eight groups (A- H).
• Confined to Africa and some time in other places including India.
• Group A is the most common form
18. Pathogenesis Mode of Transmission
• Sexual mode -most common mode of
transmission, accounts for 75% of total cases
in the world.
Heterosexual route the commonest mode.
Risk of transmission minimal (0.1- 1 % per
coitus) .
Anal intercourse - higher risk of transmission
than vaginal intercourse.
• Blood transfusion - least common mode of
transmission (5%) .
Risk of transmission is maximum (90- 95%).
20. Perinatal mode:
Risk is maximum if mother is recently infected or has already developed AIDS.
Transmission may occur at any time during pregnancy
and breast feeding bur the risk is maximum during
delivery.
In the absence of ally intervention, the risk of
transmission from mother to fetus is abour20-
40%.
21. ✓Saliva inhibitory substances like
fibronectin and gtycoproteins.
✓ casual contact or
✓ kissing .
✓ insect bite.
Viral load is
Maximum in blood, genital
secretions, and CSF.
Variable in breast milk and saliva;
zero to minimal in other body fluids
or urine.
NOT TRANSMITTED BY
22. Receptors on host cell
• Main receptor- CD4 receptor
Helper T cells ,monocytes, macrophages,
Langerhans cells, astrocytes, keratinocytes
and glial cells.
• Co-receptor - fusion into host cell.
chemokine receptors act as co-receptors
CXCR4 - on T lymphocytes.
CCR5 - macrophage lineage.
• DC-SIGN, a dendritic cell-specific lectin
receptor present in skin and mucosal
surfaces, - facilitate transport of HIV by
dendritic cells to lymphoid organs where HIV
replicates further in T cells.
23. Mutation in CCR5 (delta 32 mutation)
• This mutation results in blockade of HIV
entry into the cells.
Some lucky Europeans who are either:
• Completely resistant to HIV infection:
homozygous.
• Susceptible but progress of AIDS is
delayed: heterozygous.
24. Replication
Attachment and Fusion:
of receptor and coreceptor to gp 120 and gp 4 l.
Penetration and uncoating:
nucleocapsid enters in host cell cytoplasm,
uncoating and release ss RNA and viral enzymes.
Reverse transcription:
Viral reverse transcriptase - ssRNA into ssDNA -
DNA-RNA hybrid is formed.
RNA is degraded by viral endonuclease and ssDNA
replicates to form ds DNA.
25. Transcription of the DNA
components of viral proteins .
Pre-integration complex:
The nucleoprotein complex formed, comprises of linear dsDNA, gag matrix protein, accessory
vpr protein and viral integrase - transported into the host cell nucleus.
Integration: The viral dsDNA gets integrated in chromosome; mediated by viral integrase.
integrated virus is called as provirus.
Latency: establishes a latent infection. it is able to replicate even in latent state and is
infectious to other neighbouring cells.
26. Stages of Natural course (Immunopathogenesis)
1. Acute HIV Disease or Acute Retroviral
Syndrome
HIV is carried to lymph nodes , lymphoid tissues for
multiplication in T cells.
Destroys infected T cells
Spills over into blood stream - Primary viremia
3-6 weeks after it –
✓ Initial flu-like illness.
✓Decrease in circulating CD4 T cells.
27. 2. Asymptomatic Stage
Adequate immune response develops in 1 month.
CD8 T cells
HIV specific neutralizing antibodies
Viremia drops down
CD4 T cell count becomes normal.
State of clinical latency, but not microbiological latency.
• Persist in lymph nodes, high level viral replication.
• Clinical latency - ranges from few months to 30 years.
• If latency is broken, the disease progresses rapidly and death results in 1 - 2
years if untreated.
28. • Enlarged lymph nodes >1 cm in one or more non contiguous sites that
persist for at least 3 months.
• Replication in lymph nodes.
3. Persistent Generalized Lymphodenopathy (PGL)
29. 4. Symptomatic HIV infection (AIDS Related Complex, ARC)
Develop constitutional symptoms:
• Unexplained diarrhea lasting for more than l
month.
• Weight loss more than 10% of body weight,
fatigue malaise and night sweat
• Mild opportunistic infections such as oral thrush.
• CD4 T cell level starts falling.
30. AIDS
Characterized by:
• Rapid fall in CD4 T cell count
(usually less than 200 cells/µl)
• High virus load
• Lymphoid tissue is totally
destroyed and replaced by
fibrous tissue.
Gradually - end stage of HIV
infection called AIDS .
31. Epidemiology Global Situation
78 million people infected and
39 million deaths.
Sub-Saharan Africa
most severely affected region.
• Andhra Pradesh - worst affected ,Maharashtra
and Karnataka.
• Northeast states such as Nagaland, Mizoram and
Manipur.
Reservoir Infected people (symptomatic as well as
asymptomatic) .
32. High Risk Groups
Health care
workers (via
accidental needle
pricks or blood
splashes on eyes).
Hemophiliacs and
recipients of blood
or blood products .
33. Kinetics of Immune Response
Host immune response following infection
• Viremia: entry of virus -transient high level viremia and p24 antigenemia.
Fall down with concomitant immune response.
• Humoral response formation of antibodies lgM, IgA, lgG against different
structural proteins
gag: p15, p17, p24, p55;
env: gp 41, gp 120, gp if>O; and
pol : p31, p51 and p66.
regulatory proteins (nef, rev, cat) and
accessory proteins (vif, vpu and vpr).
34. Window period: antibodies appear in serum only after a period of interval,
which is called window period- 3 to 12 weeks.
Antibodies to gag protein (p24 and p55) usually appear first, env & pol
proteins may also be produced simultaneously.
• As infection progresses - antibody to p24 usually declines but env
persists.
Anti-HIV antibodies: lgA, lgM and IgG appear, assays detect lgG
antibodies. IgM detects early seroconversion (2- 11 days) particularly
following needle stick injury and infection in newborn.
Detection of lgA is useful in specimens such as sero mucous secretions
(saliva, colostrum, genitourinary secrecion etc.) and in newborn.
36. A . Screening Assays - Antibody Detection
ELISA - 2- 3 hours.
Rapid/simple tests - less than 30 minutes .
High sensitivity .
Should be confirmed by confirmatory tests.
• Antigens used in screening tests are:
HIV-1 specific (p24, gp 120, gp 160, gp 41)
HIV-2 specific gp 36
• They detect HIV-I and 2 either separately or together.
37. ELISA (Enzyme-linked lmmunosorbant Assay)
• Most commonly performed screening test at blood banks and tertiary care sites.
• Easy to perform, adaptable to large number of samples.
• It is sensitive, specific, and cost effective.
38. Rapid/Simple – require less than 30 minutes .
They work on various principles such as:
Dot blot assays (or lmmunoconcentration or flow through method)
Immunochromatography (or ICl; lateral flow assay)
Particle agglutination assays (using latex, gelatin, RBCs)
Dip stick/Comb tests (ELISA based assays)
39. B . Supplemental Tests
Highly specific antibody detection methods.
Western Blot - recommended by NACO.
• Immunoblot technique.
• lt detects antibody to gag gene, pol gene, env gene
products .
• The antigen antibody complexes appear as distinct
bands on nitrocellulose strip.
WHO criteria - at least two envelope bands.
CDC criteria-any two out of p24, gp 120, gpl60,
gp41 bands.
40. C. CONFIRMATORY TEST
1.Detection of p24 Core Antigen
Detectable after 1 – 2 weeks of infection and lasts for 3-4
weeks.
Again, elevated - late advanced stage of AIDS.
Less sensitive because once the antibody is formed,
antigen antibody complex gets eliminated from blood.
Recently, antigen dissociation assay
Pre-treatment of serum - liberates p24 antigen from
immunocomplexes. Better sensitivity.
41. ✓For confirmation of diagnosis of HIV /AIDS .
✓Diagnosis of HIV during window period .
✓To diagnose late stage of AIDS (immune collapse) or CNS disease.
✓Diagnosis of HIV in infants .
✓Monitoring the progress of HIV infection .
✓To resolve equivocal western blot results.
Uses of p24 antigen detection test:
42. 2. Viral RNA Detection
• “Gold standard" for confirmation
Various formats available are:
• Reverse transcriptase polymerase chain reaction
• Branched DNA assay
• NASBA: Nucleic acid sequence based amplification
• Real time RT-PCR: For estimating viral load.
43. • Routine diagnosis of HIV.
• Most sensitive and specific method, detects even few copies of viral RNA.
• Best tool -during window period.
• Viral load monitoring: Real time RT-PCR ,for monitoring ART response.
• Typing:
differentiate between HIV-I and HIV-2 infections and
can detect the specific genotype or subtype.
• Detection of drug resistance genes .
44. 3. DNA PCR
• PCR detecting proviral DNA .
• For diagnosis of paediatric HIV.
• Differentiate latent HIV infection from active viral
transcription.
• During the window period .
• Viral load estimation (real time PCR)
• Detection of genotypes.
45. 4. Isolation of the Virus from Blood or Tissues
Time consuming; expensive, and not sensitive.
Two methods are being used for virus isolation:
Viral growth in the culture supernatant
Presence of p24 antigen or reverse transcriptase enzyme or syncytia formation.
Immunofluorescence assay - viral antigens in the infected cells.
2. Co-cultivation method,
Uninfected donor cells are
stimulated with PHA and after 48-72
hours, the stimulated cells are
cultured with the PBMCs from the
patient.
1. Direct method-
Infected blood mononuclear are
cultured invitro in presence of
mitogen phytohemagglutinin
(PHA).
46. Non-specific/Immunological Tests
CD4 T cell count: Carried our by cytometry method.
Useful in:
Assessing the risk of opportunistic infections .
Initiation of antiretroviral therapy - falls below 350/mm .
Monitoring the response to antiretroviral therapy.
Abnormal proteins such as neopterin, beta 2-microglobulin and soluble ll.r2
receptor are produced by HIV activated T cells.
47.
48. Diagnosis of Pediatric HIV
The recommended methods for diagnosis of paediatric
HIV include:
• HIV DNA detection-most recommended
• HIVRNAdetection
• p24 antigen detection
• lgG ELISA only after 18 months of age
Screening methods detect lgG antibodies.
Cannot differentiate between baby's lgG or maternally transferred lgG.
Maternal antibodies - disappear by 18 months;
lgG assays can be performed after 18 months of birth
49. Control programme in India
AIDS Control Organization
NACO: has been constituted to implement the HIV/AIDS .
SACS: State AIDS Prevenition and Control Societies (SACS).
50. • NACO has formulated a strategic plan for HIV diagnosis.
Guidelines are as follows:
Situation/condition- for which the test is done .
Should use different principles or different antigens.
First screening test - the positive result should be either considered as such or confirmed
by another one or two screening tests.
1st screening test - highly sensitive
2nd and 3rd screening tests - high specificity.
• Supplememal or confirmacory tests should be used only when screening tes1t(s) results
are equivocal/ intermediate.
NACO Strategy for HIV Diagnosis
52. Clinical Diagnosis Classification systems for HIV disease
1. CDC classification system 2. WHO clinical staging of HIV/ AIDS for adults
is based on clinical conditions.
55. Principles for Selecting the First-line Regimen
(HAART) - use of combination of at least three anriretroviral
drugs.
Monotherapy - contraindicated.
NACO guideline:
First line – TLE . Single dose daily
(2NRTIs + l NNRTI) universally in all first line regimens which
is as follows:
• Choose Lamivudine in all regimen
Second line - (1 PI +2NRTIs / l NtRTI)
56. Post-exposure Prophylaxis
After needle stick injury- to reduce transnsmission
However, if started soon after exposure with in 2 hrs .
PEP can reduce the risk of HIV infection by more than 80%. .
tenofovir +lamivudine+Efavirenz single dose should be taken for 28 days.
57. NACO Guidelines to Prevent Neonatal HIV
Recommended regimen:
• Single dose of nevirapine is administered to the mother during labor and
to the baby within 72 hours after birth.
58. HIV Vaccine Strategies
Hurdles to Jump
Medical research has failed to invent an effective approved vaccine.
This attributes to various factors:
• High mutability of the virus is the single most important factor .
• Possible risk of reactivation .
• Long latent period between exposure and appearance of symptoms
• Lack of ideal small animal models for studying H rv infection
• Ethical issue: Difficulty to get human volunteers for HIV vaccine trial
• Natural immunity fails to clear HIV as it targets cells of the immune
system
• As HIV is a retrovirus: Viral genome soon gets integrated into the host
genome. Hence, it provides short window of opportunity to control .
.
59. Approaches and Trials
The researchers have conducted more than 40 vaccine trials like
• Recombinant sub-unit vaccines
• Modified envelope vaccines
• Recombinant vector vaccines
Peptide vaccines
'prime and boost' vaccines - combination of the above types of vaccines to
intensify immune responses.
BUT none of the trials has been approved for human use till now.
