1. Models Systems to Study HIV
Emily L. Lowe, Ph.D.
Microbiology, Immunology and Molecular Genetics
UCLA

2. Models Systems to Study HIV: In vitro and In vivo
In vitro: Cell lines, primary cells
In vivo: Humanized mice
In vivo: Non-human Primates

3. Models Systems to Study HIV: In vitro and In vivo
Genetically modified viruses
Modify the envelope to allow entry into more (or less) cell types
Add a fluorescent protein to enable better visualization
Green fluorescent protein (GFP), green
mCherry, red
SIV (Simian Immunodeficiency Virus)
SHIV: chimeras of HIV and SIV
Yu et al. HIV traffics through a specialized, surface-accessible intracellular compartment during trans-infection of T cells by mature dendritic cells. PLoS Pathog. 4 (8) e

4. In vitro Models to Study HIV
In vitro: Taking place in a test tube, culture dish or elsewhere OUTSIDE a living organism
Excellent tools for simple questions with limited variables
Best for testing more focused approaches
Cells are easily manipulated
Cost efficient
Scalable
Excellent for initial drug screens

5. Types of In vitro Models
Cancer cell lines are IMMORTALIZED
Can be expanded in culture for long periods of time
Are easily manipulated to express (or not) CD4, CCR5 and/or CXCR4 as well as host restriction factors
“Primary cells” come from people or animal subjects
Can purified to look at single or multiple populations
Can only be maintained in culture for short periods
Can be manipulated but not as easily as cancer cell lines

6. Examples of in vitro applications
To see HIV virions
To see an HIV infected cell infect another cell
To see where HIV goes in a cell
To see what HIV interacts with in a cell
Identify latency inducers

7. In vitro study to see HIV virions
Cryofluorescence Light Microscope
Figure 2. Cryo-Electron Microscopy (cryoEM) Imaging of Single HIV-1 Particles within a HeLa Cell by Correlative Microscopy(A) An overview differential interference contrast (DIC) image (10×) of HeLa cells cultured on an index gold electron microscopy grid and infected with HIV-1 particles pseudotyped with the envelope glycoprotein of vesicular stomatitis virus and containing GFP-Vpr.(B and C) DIC images of a selected area (red box in A) recorded with a 10× (B) or 60× (C) objective lens. Inset in C shows the raw GFP signals that were pseudocolored in red and overlaid on the DIC image in (C).(D–F) Low-dose cryoEM images of the corresponding region in the DIC image (white box in [C]) containing a single GFP-labeled virus particle at nominal magnifications of 170× (D), 3500× (E), and 50,000× (F). The boxed region corresponds to the image frame in the adjacent panel. In (F), an HIV-1 particle with a conical core (arrow) is clearly visible inside a multivesicular body. Scale bars: 50 μm in (A)–(D), 2 μm in (E), and 0.1 μm in (F).
Figure 1. Construction and Characterization of the Cryofluorescence Light Microscopy Stage(A) An overview of the cryostage (red box) mounted on an Olympus IX71 fluorescence light microscope. The three labeled tubes are for liquid nitrogen input (1), liquid nitrogen overflow protection (2), and dry nitrogen gas flow to the objective lens (3)
170X
3500X
50,000X
June et al., Direct Visualization of HIV-with Correlative Live-Cell Microscopy and Cry-Electron Tomography. Structure. 19 (11)

8. In vitro study to see HIV virions
Cryo-Tomographic Analysis of a Single Wild-Type HIV-1 Particle Bound to a HeLa Cell(A) 3D tracking (red trace) of an HIV-1 particle over a period of 28 min, superimposed on the fluorescence image of HeLa cells from a confocal series taken at the last time point.(B) A single z slice from the confocal series at the last time point overlaid with the HIV-1 GFP signals.(C) Cryofluorescence image of the identical region displayed in (B) superimposed on a cryo-differential interference contrast image recorded after plunge-freezing.(D–F) A low-dose cryo-electron microscopy image at 3500× (D) from the correlated region (circled in C), and tomographic slices (E and F) from the boxed area in (D). The upper slice (E) is near the bottom of the HeLa cell, whereas the lower slice (F), containing a virus particle, is located directly underneath the cell surface.(G–J) Four 4 nm thick tomographic slices taken at the indicated relative z height from the tomogram. In (E) and (G), arrowheads point to intracellular filamentous structures; in (E) and (F), white arrows point to the HIV-1 particle; and in (J), the arrow indicates the viral envelope proteins. Scale bars: 10 μm in (A)–(C), 1 μm in (D), and 0.1 μm in (E)–(G)
June et al., Direct Visualization of HIV-with Correlative Live-Cell Microscopy and Cry-Electron Tomography. Structure. 19 (11)

9. In vitro study to see an HIV infected cell infect another cell
Using a method to fluorescently tag HIV, researchers visualized both viral particle formation and transmission of virus from infected to uninfected T cells.  This video shows a T cell, one of the key white blood cells in the human immune system, that has been infected with HIV tagged with a green-fluorescent marker.   This infected T cell was placed in a solution of uninfected T cells (that aren’t glowing green), and high-resolution video microscopy was used to watch the infected T cell attach to an uninfected cell via a structure called a virological synapse that forms a sort of bridge between the cells and allows the virus to move directly into the uninfected cell.
Dr. Thomas Huser’s Lab

10. In vitro study to see where HIV goes in a cell it has infected
Campbell et al., Visualization of a proteasome-independent intermediate during restriction of HIV-1 by rhesus TRIM5alpha. J. Cell Biol. 180 (3)

11. In vitro study to see where HIV goes in a cell it has infected
Green = HIV
Red = “body”
Campbell et al., Visualization of a proteasome-independent intermediate during restriction of HIV-1 by rhesus TRIM5alpha. J. Cell Biol. 180 (3)

12. In vitro study to see what HIV interacts with inside a cell
Red = host restriction factor
Green = HIV
Orange = interaction!!!
Campbell et al., Visualization of a proteasome-independent intermediate during restriction of HIV-1 by rhesus TRIM5alpha. J. Cell Biol. 180 (3)

13. In vitro study to identify latency inducers
Induction of HIV gene expression in latently infected Jurkat T -cells by TNF-a. Populations of Jurkat T cells were infected with VSV-G pseudotyped vectors carrying the d2EGFP-reporter (short-lived GFP) and a wild-type T at gene. The cells spontaneously shutdown transcription and enter latency. (A) Uninfected cells. (B) Latently infected Jurkat Tcells prior to TNF-a treatment. (C) Latently infected Jurkat T -cells induced with TNF-a for 15 h. Left panels: Light micrographs of the cell population. Middle panels: Fluorescent micrographs. Right panels: Histogram of uorescent cells obtained by FACS. Arrows indicate the mean positions of the negative and positive cell populations.
Kim et al., Recruitment of THIIH to the HIV LTR is a rate-limiting step in the emergence of HIV from latency. EMBO J

14. In vitro studies have given us a lot but…
One major limitation to vaccine and therapeutic cure research has been the lack of an animal model that recapitulates all of the salient features of HIV-1 infection in humans.
In vivo: Experimentation using a whole, living organism

15. In vivo Models to Study HIV
When considering species other than human as models for HIV-1 infection, the cellular proteins of the species must support viral replication.
Humanized mice
Non-human primates

16. In vivo Models to Study HIV: Humanized Mice
Most importantly, these animals CAN be infected with HIV!
Develop systemic viremia
CD4 T cell loss

17. Akkina, R. New generation humanized mice for virus research: comparative aspects and future prospects. Virology. 435 (1)

18. In vivo Models to Study HIV: Humanized Mice
Stem cells can be genetically manipulated (gene therapy)
Not terribly cost effective
Surgery requires technical skill
Cannot be bred
Special housing/handling due to immune compromised status
Scalable allows for multiple “n”
Can make up to 30 mice per tissue pair
Excellent for initial drug-toxicity/efficacy screens
IV and mucosal routes of infection possible
Non-hematopoietic cells (lung, intestine mucosa) cannot be studied in organ systems

19. In vivo Models to Study HIV: Non-human primates
Many species of African monkeys and apes are natural hosts for SIV, but generally do not develop disease as a consequence of infection.

20. Natural Hosts (African)
SIVagm
African green monkey
Natural SIV hosts. More than 40 different African primate species are endemically infected with their own unique strain of SIV. Owing to thousands of years of virus–host co‑evolution, these natural SIV hosts typically do not develop disease as a result of their infections and are thus not useful as pathogenic models.
Similar to pathogenic lentiviral infections, SIVsmm and SIVagm infections of their natural hosts result in high levels of sustained viral replication, rapid turnover of productively infected lymphocytes (including severe depletion of mucosal CD4+ T cells) during acute infection and the activation of innate and adaptive immunity. However, these viruses do not result in chronic immune activation, progressive depletion of mucosal or peripheral CD4+ T cells, or the destruction of lymph node architecture in their respective hosts
Sharp P M , and Hahn B H Cold Spring Harb Perspect Med 2011;1:a006841
©2011 by Cold Spring Harbor Laboratory Press

21. In vivo Models to Study HIV: Non-human primates
By contrast, infection of Asian macaques, which are NOT natural hosts for primate lentiviruses, with certain strains of SIV results in high viral loads, progressive CD4+ T cell depletion and opportunistic infections.

22. Natural Hosts (African) Non-natural Hosts (Asian)
However, comparisons of non-pathogenic versus pathogenic SIV infection in African versus Asian monkeys have led to a number of important insights into AIDS pathogenesis
Sharp P M , and Hahn B H Cold Spring Harb Perspect Med 2011;1:a006841
©2011 by Cold Spring Harbor Laboratory Press

23. In vivo Models to Study HIV: Non-human primates and SIV
HIV-1 group M, which is the most prevalent HIV strain, jumped from chimpanzees into humans.
SIVcpz
HIV-1
SIVsmm
HIV-2 originated in sooty mangabeys and is responsible for fewer infections than HIV-1
Of the natural SIV hosts, SIVsmm infection of the sooty mangabey (Cercocebus atys) and SIVagm infection of the African green monkey (Chlorocebus aethiops) have been studied in detail owing to the availability of these primate species at primate centres in the United States and Europe. The sooty mangabey model is of particular interest, as cross-species transmissions of SIVsmm gave rise to HIV‑2 in humans and SIVmac in macaques.
HIV-2
SIVmac

24. In vivo Models to Study HIV: Non-human primates and SIV
SIV: Simian Immunodeficieny Virus
First isolated at the New England Primate Research Center, Massachusetts, from rhesus macaques with a transmissible form of immunodeficiency characterized by opportunistic infections and tumours
Later traced to an outbreak of lymphoma in the 1970s among macaques that were housed at the California National Primate Research Center, California
Monkeys might have received tissues from SIV-infected sooty mangabeys during experiments aiming to develop a non-human primate model for prion disease
SIVs. SIV was first isolated at the New England Primate Research Center, Southborough, Massachusetts, USA, from rhesus macaques with a transmissible form of immunodeficiency characterized by opportunistic infections and tumours109. The source of the virus was later traced to an outbreak of lymphoma in the 1970s among macaques that were housed at the California National Primate Research Center, Davis, California USA110; these macaques might have received tissues from SIV-infected sooty mangabeys during experiments aiming to develop a non-human primate model for prion disease111.

25. In vivo Models to Study HIV: Non-human primates and SHIVs
Simian immunodeficieny virus containing HIV sequences/elements
HIV-based vaccines cannot be tested with SIV
SIV is not sensitive to many drugs that inhibit HIV-1
SIV uses CD4 and CCR5 (like HIV-1) but my also use other co-receptors complicating testing of entry inhibitors
Env-SHIV
RT-SHIV
stHIV-1
SHIVs. Despite the valuable insights into lentiviral pathogenesis that have been obtained from studies of SIV infection in macaques, there are fundamental differences between SIV and HIV‑1 that limit the use of SIV–macaque models for certain applications. For example, HIV-based vaccine immunogens cannot be tested directly by challenging with SIV, and SIV is not sensitive to many of the drugs designed to inhibit the HIV‑1 protease, reverse transcriptase (RT) and integrase enzymes. In addition, although most HIV‑1 and SIV isolates use CCR5, other co‑receptors used by these viruses may differ. Whereas HIV‑1 can acquire the ability to use CXCR4, SIV rarely gains this ability, but is able to use alternative co‑receptors that are not used by HIV‑1 (REF. 119). These differences in co‑receptor use have the potential to complicate the testing of certain virus entry inhibitors in SIV-infected macaques. To address these limitations, efforts have focused on the development of SHIVs that can replicate and cause disease in macaques.

26. In vivo Models to Study HIV: Non-human primates and SHIVs
SHIVs expressing HIV‑1 Env. A number of SHIVs expressing HIV‑1 Env glycoproteins have been constructed to test Env-specific vaccines and drugs in non-human primates. Most of these recombinants were generated by replacing the rev, tat and env genes of SIVmac239 with the corresponding rev, tat, vpu and env genes of HIV‑1. Although initially these chimeric viruses replicated poorly in macaques, after extensive passage in animals they acquired the ability to replicate efficiently and cause disease. Indeed, the first generation of serially passaged SHIVs were highly pathogenic, replicating to high levels and causing rapid, nearly complete depletion of CD4+ lymphocytes as early as 3 months after infection. The prototype of this group is SHIV89.6P, which was widely used as a challenge virus until it was recognized that it is paradoxically easy to protect against by vaccination. Indeed, a range of T cell-based vaccines provide robust protection against SHIV89.6P, as measured by dramatic reductions in post-challenge viral loads relative to the loads in unvaccinated control animals, but these vaccines afford little or no protection against primary SIV isolates, such as SIVmac239 and SIVmac251. This has been attributed to several phenotypic differences between the viruses.
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio

27. In vivo Models to Study HIV: Non-human primates and SHIVs
SHIVs expressing HIV‑1 pol. One of the major limitations of SIVs and of SHIVs expressing HIV‑1 Env is that they are insensitive to many of the drugs that target HIV‑1 enzymes. To overcome this limitation, a new generation of SHIVs is being developed by substituting SIV polymerase (pol) sequences with the corresponding sequences from HIV‑1. Several of these recombinant viruses contain sequences encoding the HIV‑1 RT (FIG. 4). Unlike parental SIVs, these RT‑SHIVs are sensitive to non-nucleoside reverse-transcriptase inhibitors (NNRTIs) commonly used against HIV‑1, and treatment of RT‑SHIV-infected macaques with antiretroviral drugs effectively suppresses viraemia133,134. Importantly, these animal models have demonstrated that suboptimal treatment results in the selection of signature drug resistance mutations that are found in HIV‑1 from patients receiving antiretroviral treatment135,136. These models allow much more extensive investigations of viral replication, and recent data suggest that during highly active antiretroviral therapy (HAART), viral replication persists in multiple tissues137,138. Additional SHIVs expressing HIV‑1 protease139 or a combination of HIV‑1 RT and Env140 are also being developed.
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio

28. In vivo Models to Study HIV: Non-human primates and SHIVs
stHIV. One could envisage that the progressive substitution of SIV genes with those from HIV‑1 would eventually lead to the generation of a virus that replicates in macaques but is more closely related to HIV‑1 than to SIV. However, the opposite approach, engineering HIV‑1 to replicate in macaques, has shown more promise. Pig-tailed macaques represent an especially promising model, as they lack TRIM5 proteins that can block HIV‑1 infection. This model has been used to demonstrate the effectiveness of PrEP in providing apparent sterilizing protection with a drug cocktail commonly used against HIV‑1 in humans. Although the replication of stHIV‑1 in pig-tailed macaques is eventually controlled, it is conceivable that additional engineering to overcome other restriction factors, such as tetherin, perhaps in combination with additional passage in animals, would lead to a minimally modified HIV‑1 that can reproducibly cause disease in macaques. The development of stHIV‑1 strains might eventually allow direct efficacy testing of HIV‑1 vaccine immunogens and antiretroviral drugs in macaques.
Pig tailed macaque
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio

29. In vivo Models to Study HIV:
Non-human primates
Sign-a-mol-gous
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio

30. Pathogenesis similar to HIV-1 High viral loads
Most commonly used
Pathogenesis similar to HIV-1
High viral loads
Progressive depletion of mucosal or peripheral CD4+ T cells
Destruction of lymph node architecture
Progress AIDS faster (1-2 yrs)
Rhesus macaques. Rhesus macaques of Indian origin are by far the best characterized and most utilized non-human primate model for AIDS. The two most common SIV challenge strains, SIVmac251 and SIVmac239, were initially isolated from rhesus macaques of Indian descent and are thus particularly well adapted to these animals, with infections resulting in high viral loads with minimal animal-to‑animal variation (TABLE 1). Similarly to HIV‑1 replication, ongoing SIV replication results in the turnover and progressive loss of CD4+ T cells, particularly in the GALT74. However, the rate of disease progression for SIV-infected animals is considerably more rapid than for HIV‑1‑infected humans; Indian-origin rhesus macaques typically progress to AIDS within 1–2 years of SIV infection, compared with 8–10 years for humans who are infected with HIV‑1 and not receiving antiretroviral therapy.
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio

31. Next most commonly used
Lower viral loads and less CD4+ T cell depletion
Progress AIDS faster (within 42 weeks)
Pig-tailed macaques. Next to rhesus macaques, pig-tailed macaques are the most commonly used non-human primate model for AIDS. Although viral loads and the rate of CD4+ T cell depletion do not differ substantially in pig-tailed versus rhesus macaques (TABLE 1), pig-tailed macaques progress to disease more rapidly92,93. On average, pig-tailed macaques develop AIDS within 42 weeks of SIV infection compared to 70 weeks for rhesus macaques.
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio

32. Least used
SIV strains are less pathogenic (probably because they are passaged in Rhesus macaques)
(Sign-a-mol-gous) Cynomolgus macaques. Cynomolgus macaques have not been used as widely in AIDS research as rhesus or pig-tailed macaques, in part owing to historical reasons pertaining to SIV isolation and the less extensive immunogenetic characterization of this species. A comparison of viral loads and CD4+ T cell turnover revealed that SIVmac251 and SHIV89.6P are generally less pathogenic in cynomolgus macaques than in Indian or Chinese rhesus macaques88 (TABLE 1). For Chinese rhesus macaques, these differences probably reflect, at least to some extent, the incomplete adaptation of these viruses to this species owing to their passage history in Indian rhesus macaques.
Hatziioannou, T. and Evans, D. T. Animal Models for HIV/AIDS Research. Nat. Rev. Microbio

33. In vivo Models to Study HIV: Non-human primates
Stem cells can be genetically manipulated (gene therapy)
Cost prohibitive
Primate research contains ethical controvery
Not scalable
IV and mucosal routes of infection possible
All tissues match (non-hematopoietic and immune) allowing for organ systems to be studied

34. Models to study HIV Cats and Feline Immunodeficiency Virus
Wongsrikeao et al., Antiviral restriction factor transgenesis in the domestic cat. Nature Methods

35. Thank you!