**3. Defining animal models**

#### **3.1. Macaques**

reported in the USA with an immune deficiency syndrome otherwise known as simian AIDS has been termed STLV-III mac. It is worthy to note that STLV types I and III are similar and/ or related to the human T lymphotropic viruses (HTLV-I and LAV/HTLV-III) the causative agent(s) for AIDS. The striking similarities include growth characteristics, similar size of viral structural proteins, morphology, T4 cell tropism and serological cross reactivity of viral proteins [21]. The residing proteins found in the simian virus also have similar molecular weight with respect to the gag and env encoded proteins of LAV/HTLV-III [22]. Both are recognized by reference LAV/HTLV-III human serum and monoclonal antibodies to the core protein, p24, of the human virus. These proteins are basic and have relevant information needed in the development of candidate vaccine, rapid diagnostics and elucidation of HIV virology [21]. Knowledge of the molecular structure and pathobiology of simian viruses yielded a wealth of

162 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

information and was very useful in the study of the HIV and AIDS in humans [21].

In addition to looking for other lentiviruses, rodents were genetically engineered so that their cells could express both the human version of the CD4 receptor as well as the chemokine co-receptors to which HIV-1 binds, notably the main route of entry to target cells [23]. The envelope glycoprotein 120 (gp120) domiciled on the surface of the HIV-1 virus fuses with the host target cell membrane specifically invoking a cascade of activity involving the CD4+ receptors and chemokines co-receptors thus initiates viral entry. In successfully developed transgenic mice, however, the gp120 will not successfully bind to CD4-expressing T cells thus preventing targeted cell infection. Replacement of the gp120 coding region of the HIV with gp80 region obtained from the murine leukemia virus results in altered virus thus overcoming the problem. This chimeric HIV-1 clone could infect conventional mice cells, but not human cells. Although this has been extensively adopted in research, these models could not produce some disease progression seen in humans, for

Humanized mice model also known as humice are mice carrying functioning human genes, cells, tissues, and/or organs engraftment mostly on genetically modified mouse background. They replicate the human HIV immune responses and are currently used to study mechanisms of immune activation, mucosal transmission and prevention, immune pathogenesis

The development of fitting animal models is seen as one of the most important challenges in studies of co-infection, since HIV does not cause disease in rodents and in non-human

Lentiviruses specific to other species that also compromise the immune systems in ways similar to HIV-1 have been useful in providing information about the pathogenicity of the virus. In spite of the similarities to HIV, there are species-specific differences in their respective gene products as well as the pathogenesis of the disease fueling the drive for search of better mod-

**2.3. In rodents**

example, neuro-HIV disease [9].

and anti-viral drug development.

primates [24].

els of HIV disease control.

**2.4. Developmental perspectives of HIV animal models**

SIV in macaques follow a disease course that is similar to HIV in humans. This is useful since it can be exploited for evaluation of drug/vaccine candidates closely related to that being developed for respective human HIV infection. The model thus provides leading and insightful results in and related to drug safety and efficacy of prospective candidates [1]. Certain animal models have been developed over the years through intensive research for insightful and revealing studies on HIV/AIDS and associated cancers. These include both specific rat and mouse models designed for HIV pathogenesis and candidate vaccine development. Scientists have albinitio created the SIV non-human primate(NHP) model (**Figure 1**), for example, the Indian-origin rhesus macaque (*Macaca mulatta*), *Cynomolgous* macaque (*M. fascicularis*), and pigtailed macaque (*M. nemestrina*), for same purposes including development of microbicides. These models are useful in elucidating the mechanism of AIDS pathogenesis as well as in preclinical testing of novel drugs directed at HIV infection and cancer [1, 25, 26].

SIV infection in macaques has been used as a model for AIDS since it was established that non-human primates are resistant to infection by HIV (**Table 1**). Simply put, SIV is a retrovirus causing immunodeficiency similar to AIDS in Asian macaques. More importantly, Macaques also develop TB that is very similar to that of humans, other notable similarities in viral activity and disease manifestation include cavitary lung disease and necrotic lession. The TB latency seems in contrast to humans to have only a small proportion of lately infected Macques develop reactivation [27] though it develops persistent Mycobacterium *bovis bacillus Calmette Guerin* (BCG) [28] and *M. tuberculosis* co-infection [29].

**Figure 1.** Historical trend and impact of HIV/AIDS research in NHPs.


**Animal model**

**Cons**

•

High cost, maintenance and

• •

Identity, dose, and route of virus challenge known.

•

Steady environmental conditions can be applied for long periods

therefore enhancing the chances to detect genetic effects

Varying environmental settings can be imposed sequentially on

NHP in order to characterize genotype-environment interactions;

Possibility of testing genetic hypotheses in a prospective selective

Important and needed invasive and terminal experiments can be

Control for various clinical parameters that are

virtually impossible to control in humans (time of

•

infection, duration of ART etc).

availability.

•

Have limitated suitability for

vaccine studies.

•

SIV differs from HIV in genetic

•

Comprehensive cellular and anatomic characterization of both active and persistent reservoirs (includ-

• mating

• conducted and validated

organization especially the Vpx

gene of SIV and Vpu of HIV

ing elective necropsy).

• within 6–12

months while the

human AIDS develops after several years of infection with HIV

**Table 1.**

Advantages and disadvantages of non-human primate models used in HIV-1 research.

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is possible.

•

Testing of "risky" interventions (i.e., cell depletion

experiments, stem cell-based interventions etc).

Simian AIDS generally develops

•

Pilot trials of *in vivo* eradication conducted in a

timely and controlled fashion; treatment interruption

**Species** **Genetic comparison of non human primates in HIV research**

**Opportunities provided by the NHP models in studies** 

**Pros**

**of HIV eradication**

**Cons**

**Pros**

**Retroviral study sub-types**


**Animal model**

Macaques

Rhesus

•

Resistant to HIV.

which Macaques are resistance to especially with

respect to the association of TB reactivation and viral

SIV shows discrepancies to HIV

macaques

(*Macaca mulatta*)

load.

• • • •

Pigtail

•

SIVmac infections typically does not refelct HIV-1

•

In the female vaginal ecology,

physiology and intravaginal virus

challenge is similar to that of

human.

•

SIV/SHIV and STI co-infection

models can be studied.

infection and is more aggressive.

Usually more expensive to maintain

Not an established model for evaluation of vaccine

macaques (*Macaca* 

*nemestrina*

• • candidates

Cynomologus

•

Difficult in sample collection due to smaller size.

• to handle.

• • • PBMC.

Supports HIV-2 replication but not HIV-1

Baboon microglial cells can be infected by SHIV chimera with

strong tropism for baboon PBMC but not for rhesus macaque

It is more readily available.

Has smaller size therefore, easier

macaques

•

Exhibit low viral loads.

(*M. fascicularis*)

Baboons

• • •

Not a good model for heterosexual trans

mission as shown in SHIV89.6P chimera model

Its suitability for vaccine studies is low.

Limited HIV replication activity in monocytes or

macrophages, CSF or brain

Availability of some rhesus macaques depends on

domestic breeding capacity and skill.

Have poorly characterized MHC allelic profiles and

may not be suitable for vaccine studies.

May not be appropriate for comparative menstrual

cycle-related SIV/SHIV studies.

Low turnaround of the model.

**Species**

**Cons**

**Pros**

•

Can be infected with SIV which

SIVmac239

SIVsmPBj6.9

SIVsmPBj6.6

SIVPBj14

SIVmac316

SIVsmE543–3

SIVsmE660

SIVmac251

164 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

RT-SHIV

SHIV-SF162P3

SIVmac251 +

SHIV/17E-Fr

SIVmne

SIVmneCl8

SIVmne170

SIVmne027

RT-SHIV

SIVmac251

compromises immunity

•

Ability to causes secondary

complications similar to HIV in

humans.

•

Intravenous, intrarectal, intravaginal and penile-exposure models

are established

• models.

•

Indian macaques have well characterized MHC allelic profiles thus

suitable for as a model for vaccine

candidates.

Studies of SIV/TB co-infection

**Retroviral study sub-types**

**Table 1.** Advantages and disadvantages of non-human primate models used in HIV-1 research. In the model, co-infection with BCG and SIV hastened the progression to AIDS [30] and reveals severe diminution of CD4+ T cells, loss of BCG-specific T cell responses, and reactivation of the clinically latent BCG infection into a TB-like disease as reported by Shen et al. [31]. *M. tuberculosis* reactivation in SIV-infected macaques is linked with peripheral T-cell depletion instead of viral load [32].

*3.3.1. Murine AIDS*

In many ways, murine AIDS (or MAIDS) and human AIDS are similar. Immunological analysis and genetic studies reveal resistant gene(s) in the H-2 complex of mice, an indication that genetic differences in mice could modify features of HIV disease. The defective murine leukemia virus is the major etiologic agent of MAIDS, which seems to be able to induce disease in the absence of virus replication. Target cell proliferation and oligoclonal expansion are induced by the virus, which suggests repressed immunity seen in mice thus referred to as paraneoplastic syndrome. This is further supported by the good response(s) of MAIDS mice to antineoplastic agents. This animal model is useful in demonstrating the emergence of novel hypotheses about AIDS, including the roles of defective HIV and HIV replication in the progression of the disease, and also the importance of identifying the HIV targeted cells *in vivo*. Although MAIDS and AIDS are triggered by retroviruses of different classes, the availability of a model in small, accessible animal species with elaborated genetics is beneficial in understanding the pathogenesis of AIDS especially in cases where one or more of the affected cel-

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Potash et al. [40] designed a model of HIV-1 infection of mice for the study of viral replication, its pathogenesis and control. The team substituted the coding region of gp120 in HIV-1/ NL4–3 with gp80 from ecotropic murine leukemia virus, which infects only rodents, targeted at infecting rodents with HIV-1 in rodents. The EcoHIV was developed through the chimeric virus construct, which productively infected lymphocytes in mice, but failed to do the same in human lymphocyte culture. It was recorded that immunocompetent adult mice were easily prone to infection by a single dose EcoHIV inoculant as the demonstrated by viral detection in lymphocytes in the spleen, brain cells and peritoneal macrophages. The passage in culture, and induction of antibodies to HIV-1 Gag and Tat showed that the animal produced virus was

Mice are not susceptible to HIV infection due to the virus specificity for the human cell. These would have otherwise been ideal models, however, owing to the large diverse tools and wide knowledge about the rodent immunity. To circumvent limitation in mice (**Table 3**), complementary mouse models have thus been developed over the years targeting specific genes (**Table 4**). Using these models, the more important features of HIV infections and *M. tuberculosis* can be replicated in mice (e.g. virus replication in splenic lymphocytes, peritoneal macrophages and brain tissue; typical TB granuloma formation; immune repression and/or chronic immune stimulation; and susceptibility to systemic, vaginal, and rectal infection by HIV) [24]. Mice modified genetically are often used for research and/or simply as an animal model of human diseases. The use of genetic engineering tools has greatly improved the ability to develop various mouse models important to preclinical research. With the recent developments in gene editing technologies, it is now possible to generate quickly highly adjustable

lular and molecular pathways are common in both diseases [39].

*3.3.2. Genetic modifications/gene manipulations*

indeed infectious and immunogenic, respectively.

*3.3.3. Transgenic mice*

#### **3.2. Other primates**

Chimpanzees support productive infection, but the disease does not occur for at least 10 years. Alter et al.'s [33] investigative study was designed to determine the possibility of using a transmissible agent in humans with capability to induce AIDS in non-humans thus established an animal model in which the pathogenesis, treatment regimen, and prevention of AIDS could be studied (**Table 1** and **Figure 1**). This early attempt pre-dates the virologic investigations that linked human AIDS to a type C retrovirus [33]. The NHP models have recorded tremendous successes, the limitations observed notwithstanding (**Table 2**).

Interestingly, Baboons can support replication of certain strains of HIV-2, but difficult with HIV-1 strains (**Table 1**). It has been shown that HIV infection replicates mainly in the T-cells, with limited or no activity in the monocytes or macropahges, CSF or brain of Baboons and macaque monkeys [34–36].

#### **3.3. Mice**

For better assessment of the HIV-linked clinical presentations, murine models have been developed and proved a better tool in elucidating the mechanism of disease progression. Equally giving lead to scientific direction as against non-human primates [37] geared toward the future of HIV drug and vaccine development [38]. Besides that, it is usually costly to work with the non-human primates (large animals), which further underscores the necessity for murine models [9].


**Table 2.** Successful clinical testing conducted in NHP models.

#### *3.3.1. Murine AIDS*

In the model, co-infection with BCG and SIV hastened the progression to AIDS [30] and reveals severe diminution of CD4+ T cells, loss of BCG-specific T cell responses, and reactivation of the clinically latent BCG infection into a TB-like disease as reported by Shen et al. [31]. *M. tuberculosis* reactivation in SIV-infected macaques is linked with peripheral T-cell deple-

166 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

Chimpanzees support productive infection, but the disease does not occur for at least 10 years. Alter et al.'s [33] investigative study was designed to determine the possibility of using a transmissible agent in humans with capability to induce AIDS in non-humans thus established an animal model in which the pathogenesis, treatment regimen, and prevention of AIDS could be studied (**Table 1** and **Figure 1**). This early attempt pre-dates the virologic investigations that linked human AIDS to a type C retrovirus [33]. The NHP models have

Interestingly, Baboons can support replication of certain strains of HIV-2, but difficult with HIV-1 strains (**Table 1**). It has been shown that HIV infection replicates mainly in the T-cells, with limited or no activity in the monocytes or macropahges, CSF or brain of Baboons and

For better assessment of the HIV-linked clinical presentations, murine models have been developed and proved a better tool in elucidating the mechanism of disease progression. Equally giving lead to scientific direction as against non-human primates [37] geared toward the future of HIV drug and vaccine development [38]. Besides that, it is usually costly to work with the non-human primates (large animals), which further underscores the necessity for

adverse effects not found using short term-

HIV infection as seen in the treatment for

infection with SHIV however, they did have

Tenofovir

Various

MRK-Ad5

Tenofovir and AZT

high dose treatment

Macaques The effectiveness of prophylaxis in blocking

Macaques Provided guidelines for antiviral treatment in HIV positive pregnant mothers

Macaques The Monkeys were not protected against

lower viral

occupational exposures.

**Evaluation Species Study outcome Drug**

Efficacy and toxicology Macaques Long term-highdose HIV treatments had

recorded tremendous successes, the limitations observed notwithstanding (**Table 2**).

tion instead of viral load [32].

macaque monkeys [34–36].

**3.3. Mice**

murine models [9].

Prophylactic treatment with

Mother-to-fetus transmission, and

Vaccine efficacy in SHIV 89.6p, a hybrid SIV, genetically engineered

**Table 2.** Successful clinical testing conducted in NHP models.

anti-virals

from HIV

fetal prophylaxis

**3.2. Other primates**

In many ways, murine AIDS (or MAIDS) and human AIDS are similar. Immunological analysis and genetic studies reveal resistant gene(s) in the H-2 complex of mice, an indication that genetic differences in mice could modify features of HIV disease. The defective murine leukemia virus is the major etiologic agent of MAIDS, which seems to be able to induce disease in the absence of virus replication. Target cell proliferation and oligoclonal expansion are induced by the virus, which suggests repressed immunity seen in mice thus referred to as paraneoplastic syndrome. This is further supported by the good response(s) of MAIDS mice to antineoplastic agents. This animal model is useful in demonstrating the emergence of novel hypotheses about AIDS, including the roles of defective HIV and HIV replication in the progression of the disease, and also the importance of identifying the HIV targeted cells *in vivo*.

Although MAIDS and AIDS are triggered by retroviruses of different classes, the availability of a model in small, accessible animal species with elaborated genetics is beneficial in understanding the pathogenesis of AIDS especially in cases where one or more of the affected cellular and molecular pathways are common in both diseases [39].

#### *3.3.2. Genetic modifications/gene manipulations*

Potash et al. [40] designed a model of HIV-1 infection of mice for the study of viral replication, its pathogenesis and control. The team substituted the coding region of gp120 in HIV-1/ NL4–3 with gp80 from ecotropic murine leukemia virus, which infects only rodents, targeted at infecting rodents with HIV-1 in rodents. The EcoHIV was developed through the chimeric virus construct, which productively infected lymphocytes in mice, but failed to do the same in human lymphocyte culture. It was recorded that immunocompetent adult mice were easily prone to infection by a single dose EcoHIV inoculant as the demonstrated by viral detection in lymphocytes in the spleen, brain cells and peritoneal macrophages. The passage in culture, and induction of antibodies to HIV-1 Gag and Tat showed that the animal produced virus was indeed infectious and immunogenic, respectively.

#### *3.3.3. Transgenic mice*

Mice are not susceptible to HIV infection due to the virus specificity for the human cell. These would have otherwise been ideal models, however, owing to the large diverse tools and wide knowledge about the rodent immunity. To circumvent limitation in mice (**Table 3**), complementary mouse models have thus been developed over the years targeting specific genes (**Table 4**). Using these models, the more important features of HIV infections and *M. tuberculosis* can be replicated in mice (e.g. virus replication in splenic lymphocytes, peritoneal macrophages and brain tissue; typical TB granuloma formation; immune repression and/or chronic immune stimulation; and susceptibility to systemic, vaginal, and rectal infection by HIV) [24].

Mice modified genetically are often used for research and/or simply as an animal model of human diseases. The use of genetic engineering tools has greatly improved the ability to develop various mouse models important to preclinical research. With the recent developments in gene editing technologies, it is now possible to generate quickly highly adjustable


**Table 3.** Comparison of major mice chimeric models used in HIV-1 research.

mouse models tailored to research needs. Mouse is still putatively the preferred animal model used in drug discovery and therapeutic agent development [52]. Below are some specific examples of genetically modified mouse model backgrounds which resulted from targeted mutations of specific mouse genes as presented in **Table 4**.

> Earlier versions of humanized mice were developed mainly to study HIV-1 infection especially in modeling for immune-pathogenesis [53, 54], although the SCID-hu Thy/Liv model is still used to test for antiviral drugs [55]. In the improved humanized mice strains, several HIV-1 strains have been successfully used for HIV infection in the developed mouse model, and these include CCR5-tropic [56], CXCR4-tropic and dual-tropic (NL4-R3A) viruses [56]. Obviously, HIV-1 infection can be established in immune-deficient mouse models by inoculation through various routes of entry, namely intraperitoneal, intravenous and/or mucosal routes [57, 58]. Various research reports have established sustained viral replication and

**Table 4.** Common genetic mutations found in mouse models and their functions (source: Ibeh et al. [52]).

*decreased innate responses especially when combined with Myd88 mutation.*

This is required for normal expression of major histocompatibility class I proteins which displays viral and self-antigens to responsive T cells and secondly for CD8+ T cell

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*Foxn1nu* mutation is generally known as nude mutation. Homozygote (*nu/nu)* type lack a thymus that is they are 'hypothymic'/'athymic' and thus are T cell deficient. Their responses to thymus-dependent antigens are poor. However, the allogenic and xenogenic grafts

*The Il2rg* is required for IL2, IL4, IL7, IL9, IL15, and IL21 high-affinity binding and signaling. It is required in mediating susceptibility to thymic lymphomas in mice. Mostly observed is the *Il2rg* deficiency that blocks the development of NK cells and the resultant defects in

transcription factor NF-κB in innate immunity signal transduction. Myd88 mutation leads

*Prf1* is a pore-forming protein that is an important component of the lytic pathway by

The *scid* mutation in the *Prkdc* gene means severe combined immunodeficient. *Prkdc* plays a role in repairing double-stranded DNA breaks and in recombining the variable (V), diversity (D), and joining (J) segments of immunoglobulin and T-cell receptor genes. Homozygous (scid/scid) mutants have no mature T and B cells, cannot mount cell-mediated and humoral adaptive immune responses, do not reject allogeneic and xenogeneic grafts, and are useful cancer research models. The disadvantage is its leakiness as some functional B and T cells as they age, in non-SPF conditions. They cannot be as thoroughly irradiated as other immunodeficient models before being engrafted renders NOD mice diabetes-free.

*Rag1* is essential for the V(D)J gene rearrangements that generate functional antigen receptors in T and B cells; homozygous *Rag1tm1Mom* mutants have no mature, functional T and B cells. The *Rag1tm1Mom* mutation on the NOD background renders NOD mice diabetesfree. However, aging NOD.129S7(B6)-*Rag1tm1Mom*/J mice develop B cell lymphomas at a high

Toll-like receptor adaptor molecule 1. It is an adapter protein used by TLR 3 to activate transcription factor NF-κB in innate i immunity signal transduction*. Its mutation leads to* 

Myd88 is critical adaptor protein utilized by all TLRs (except TLR 3) to activate

maturation and NK cell development.

innate immunity.

though may have NK activity show evidence of leakiness.

to decreased *innate responsesespecially neutrophils, macrophages, hematopoietic, molecular signaling, and apoptotic abnormalities.*

which NK and CD8+ lymphocytes kill targeted cells.

Greatly increased susceptibility to infection.

depletion of CD4+ T-cell using the routes of infection.

frequency.

*Gene name* **Characteristics**

*B2m*

*Foxn1*

*Hfh11*

Il2rg

Myd88

*Prf1* perforin 1

*Prkdc*

*Rag1*

*gene 1*

*Ticam1 Trif*

*beta-2 microglobulin*

*forkhead box N1, formerly* 

*interleukin 2 receptor, gamma chain*

*myeloid differentiation primary response gene 88*

protein kinase, DNAactivated, catalytic polypeptide

*recombination activating* 

#### *3.3.3.1. NOD/SCID mice*

Since the early 2000s, a series of immune-deficient mice suitable for developing humice have been successively designed through the introduction of IL-2Rγnull gene (e.g. NOD/SCID/ γcnull and Rag2nullγcnull mice) using various genomic approaches. Mouse backgrounds serve as the basic genetic modified rodent from which other disease models are generated mostly by further modification and/or by human tissue engraftment. These mice were generated by genetically introducing human cytokine genes into NOD/SCID/γcnull and Rag2nullγcnull mouse backgrounds [52]. There are other models that rely on the transplantation of human tissues into the SCID mice, and they are referred to as the SCID-hu mouse model.


**Table 4.** Common genetic mutations found in mouse models and their functions (source: Ibeh et al. [52]).

mouse models tailored to research needs. Mouse is still putatively the preferred animal model used in drug discovery and therapeutic agent development [52]. Below are some specific examples of genetically modified mouse model backgrounds which resulted from targeted

**Parameters HIV/SCID-hu HIV/hu-HSC HIV/BLT Reference**

Small Different same Similar Yes Yes Yes Similar Minimal NOD/ NSG mice irradiated and injected with human HSCs 2–3 months T & B cells, DCs

Small

Yes Similar Minimal

HSCs 5–7 months T & B cells, monocytes, macrophages, NK cells, DCs Murine lymph organs, rectum, vagina, gut, bone marrow Over 12 months

Different same Similar Yes Yes

NOD/SCID mice irradiated & implanted with fetal human thymus/live plus injection with

[41] [42–44] [45–48] [46, 49] [47, 50, 51]

Murine lymph organs and bone marrow 6–7 months

Small

168 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

Different same Similar Yes Yes Yes Similar Minimal SCID mice implanted with fetal human thymus/live. 5–7 months from time of birth T cells

Only thymus/live implant

Grafts last almost 12 months

Since the early 2000s, a series of immune-deficient mice suitable for developing humice have been successively designed through the introduction of IL-2Rγnull gene (e.g. NOD/SCID/ γcnull and Rag2nullγcnull mice) using various genomic approaches. Mouse backgrounds serve as the basic genetic modified rodent from which other disease models are generated mostly by further modification and/or by human tissue engraftment. These mice were generated by genetically introducing human cytokine genes into NOD/SCID/γcnull and Rag2nullγcnull mouse backgrounds [52]. There are other models that rely on the transplantation of human tissues

into the SCID mice, and they are referred to as the SCID-hu mouse model.

mutations of specific mouse genes as presented in **Table 4**.

**Table 3.** Comparison of major mice chimeric models used in HIV-1 research.

*3.3.3.1. NOD/SCID mice*

**Animal Model**

Mice • Sample size

humans

to HIV

regimens etc. • Ability to deplete major immune components • Reservoir comparison to human/HIV

• Anatomical comparison with

• Similarity of infective agent

• Infection manifestation in comparison with human/HIV • Availability for experimental infection in controlled conditions vis-à-vis route and dose of virus inoculation, drug

• Cost of maintenance compared to NHP • Methods mouse model development

• Timeframe needed for mouse

• Cellular composition during

development

reconstitution • Degree of colonization • Length infection sustained

> Earlier versions of humanized mice were developed mainly to study HIV-1 infection especially in modeling for immune-pathogenesis [53, 54], although the SCID-hu Thy/Liv model is still used to test for antiviral drugs [55]. In the improved humanized mice strains, several HIV-1 strains have been successfully used for HIV infection in the developed mouse model, and these include CCR5-tropic [56], CXCR4-tropic and dual-tropic (NL4-R3A) viruses [56]. Obviously, HIV-1 infection can be established in immune-deficient mouse models by inoculation through various routes of entry, namely intraperitoneal, intravenous and/or mucosal routes [57, 58]. Various research reports have established sustained viral replication and depletion of CD4+ T-cell using the routes of infection.

Evidently, Nie et al. have shown similar depletion of CD45RAþ naive and CD45RA + effector/ memory CD4þT lymphocytes by CXCR4- tropic HIV-1 in humanized mouse as were observed in HIV-1 patients. Similarly, the preferential depletion of CD45RA + CD4þT lymphocytes by CCR5-tropic HIV-1 was also observed. Further reports on humanized mice have shown its usefulness as a tool for studying various aspects of HIV-1 infection namely the roles of regulatory T cells (Tregs) [57], dendritic cells (pDCs) revealing the pathophysiology of human DC subsets [59] and pDC instigator function during disease initiation [60], HIV-1 immuno-pathogenesis [54, 61, 62], development of new antiviral therapy [6, 63], mucosal transmission, microbicide development [64] and currently in studying latent HIV infections. Latent HIV infections can now be established in a mouse model in the presence of administered ARV [65, 66].

HIV-susceptible human CD4þT cells, as well as other relevant populations [67]. Similarly, Dagur et al. in a current study demonstrated dual reconstitution in TK-NOG mouse model as

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The human hematopoietic progenitor cells (CD34+) from human cord blood are used to reconstitute the immune system of immune-deficient mice also known as humanized mouse [41]. An additional feature or rather advancement incorporates a fragment of the fetal human thymus engraftment, which performs functionally as a human thymus. The significance of this is to allow for a more proper positive/negative T-cell selection previously not obtainable from the original model [74]. Immunologi and virologic parameters such as CD4+ cell depletion, extent of viremia, and co-receptor-mediated tropism were all observed in HIV infection of humanized experimental mice [74, 75]. The humice demonstrated transplanted human cells in mucosal linings therefore, most possibly get infected by intravaginal and/or intrarectal routes [76]. This model is used to evaluate novel approaches in HIV prevention and treatment options including human-neutralizing antibodies, usage of prophylactic anti-retroviral therapies, and T cell-specific siRNA transfer [77]. The effect of M. tuberculosis infection on the induction of HIV gene expression has been studied with HIV transgenic mice integrating the entire viral genome [78]. In this model, viral gene expression was triggered by M. tuberculosis

The question of whether or not there should be a standardized model is the basis on which the current controversies in HIV research rest on [26]. Differences in SIV and SHIV replication in the rhesus macaque, cynomolgous and pigtailed macaques' species have been observed and is favored in the design of experimental models depending on the question raised [40]. For vaccine research, the rhesus does present the ideal for pathogenesis research; however, demanding for its use as a standard does present problems the current wave is to base considerations on the transgenic mice models. Besides, vaccine testing in more than one species of macaques with similar vaccine modalities provides an opportunity to compare outcomes

Regulatory authorities require vaccine candidates to undergo preclinical evaluation in animal models before they enter the clinical trials in humans [80]. The overarching goal of a new vaccine is to stimulate the immune system to elicit an effective immune response against the pathogen it has been designed for, and currently no alternatives to live animal use currently exist for evaluation of this response despite advances in computational sciences for the search

Integral studies such as elucidation of immune protection mechanism, optimizing route and constitutions of vaccines; determining the onset and duration of immunity, as well as satisfying safety and efficacy requirements of the new vaccines, must be done in an integrated

a possible platform to investigate hepatocyte-related HIV-1 immunopathogenesis [73].

and suppressed after anti-mycobacterial chemotherapy [78].

thus increasing confidence of research reproducibility [79].

**4. Model suitable for vaccine trials**

of an *in-silico* model [80].

*3.3.3.3. Other examples*

#### *3.3.3.2. BLT mice*

Consequently, latency has been successfully generated in humanized BLT mice [67, 68]. Available report has shown that poly lactic-co-glycolic acid (PLGA) nanoparticles with encapsulated rilpivirine (an anti-retoviral drug) coated reproductive tract offered significant protection to BLT humanized mouse model from a vaginal high-dose HIV-1 challenge [68]. Several improvements of humice models with an enhanced human immune cell reconstitution especially the female genital tract tissues create a potential mice, susceptible to intravaginal HIV infection. This type of mouse model will enable studies on mechanisms involved in HIV transmission *in vivo* and represent powerful tool for studying hematopoiesis, inflammatory disease and viral host-pathogen interactions. Several potent HIV vaccines have been put on trial and enjoyed a well-publicized but prematurely terminated results due to high frequency of seroconversions among vaccine recipients [69, 70]. Previously, the only known model for HIV testing is infection of rhesus macaques with simian immunodeficiency virus (SIV) which has provided an excellent non-human primate model for studying HIV pathogenesis [71]. This model, however, has three major disadvantages despite its application in transmitting HIV experimentally to rhesus macaques across the cervicovaginal or rectal mucosa. The established scenario makes it possible to test for microbicides and engages in laboratory study of mucosal HIV transmission. First, they are costly both in procurement and housing (limited number of primate facility globally) and is in high demand; secondly, SIV differs from HIV in genetic organization especially the Vpx gene of SIV and Vpu of HIV and lastly, while simian AIDS generally develops within 6–12 months of infection, the human AIDS develops after several years of infection with HIV (**Table 1**). However, these limitations serve as impediments in the search for an appropriate model of HIV drug testing and disease study, the transgenic mice model has overcome these feared problems associated with the SIV model. Mice have the utmost advantages of being inexpensive, have high reproductive capacity and may be housed in large numbers in a fairly small facility [72]. Furthermore, conduct of experimentation can be done in large numbers and in replicates. The severe combined immune deficiency mouse engrafted with human peripheral blood mononuclear cells (hu-PBL-SCID) could be co-engrafted with xenografts containing the dual of human fetal thymus and liver tissue (SCID-hu thy/liv)/(SCID-hu thy/liv), and this model is widely applied in preclinical evaluation of antiretroviral therapy [55, 58]. In another study, Denton et al. supported these findings and showed that the female reproductive tissues in BLT mice are adequately reconstituted with HIV-susceptible human CD4þT cells, as well as other relevant populations [67]. Similarly, Dagur et al. in a current study demonstrated dual reconstitution in TK-NOG mouse model as a possible platform to investigate hepatocyte-related HIV-1 immunopathogenesis [73].

#### *3.3.3.3. Other examples*

Evidently, Nie et al. have shown similar depletion of CD45RAþ naive and CD45RA + effector/ memory CD4þT lymphocytes by CXCR4- tropic HIV-1 in humanized mouse as were observed in HIV-1 patients. Similarly, the preferential depletion of CD45RA + CD4þT lymphocytes by CCR5-tropic HIV-1 was also observed. Further reports on humanized mice have shown its usefulness as a tool for studying various aspects of HIV-1 infection namely the roles of regulatory T cells (Tregs) [57], dendritic cells (pDCs) revealing the pathophysiology of human DC subsets [59] and pDC instigator function during disease initiation [60], HIV-1 immuno-pathogenesis [54, 61, 62], development of new antiviral therapy [6, 63], mucosal transmission, microbicide development [64] and currently in studying latent HIV infections. Latent HIV infections can

170 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

now be established in a mouse model in the presence of administered ARV [65, 66].

Consequently, latency has been successfully generated in humanized BLT mice [67, 68]. Available report has shown that poly lactic-co-glycolic acid (PLGA) nanoparticles with encapsulated rilpivirine (an anti-retoviral drug) coated reproductive tract offered significant protection to BLT humanized mouse model from a vaginal high-dose HIV-1 challenge [68]. Several improvements of humice models with an enhanced human immune cell reconstitution especially the female genital tract tissues create a potential mice, susceptible to intravaginal HIV infection. This type of mouse model will enable studies on mechanisms involved in HIV transmission *in vivo* and represent powerful tool for studying hematopoiesis, inflammatory disease and viral host-pathogen interactions. Several potent HIV vaccines have been put on trial and enjoyed a well-publicized but prematurely terminated results due to high frequency of seroconversions among vaccine recipients [69, 70]. Previously, the only known model for HIV testing is infection of rhesus macaques with simian immunodeficiency virus (SIV) which has provided an excellent non-human primate model for studying HIV pathogenesis [71]. This model, however, has three major disadvantages despite its application in transmitting HIV experimentally to rhesus macaques across the cervicovaginal or rectal mucosa. The established scenario makes it possible to test for microbicides and engages in laboratory study of mucosal HIV transmission. First, they are costly both in procurement and housing (limited number of primate facility globally) and is in high demand; secondly, SIV differs from HIV in genetic organization especially the Vpx gene of SIV and Vpu of HIV and lastly, while simian AIDS generally develops within 6–12 months of infection, the human AIDS develops after several years of infection with HIV (**Table 1**). However, these limitations serve as impediments in the search for an appropriate model of HIV drug testing and disease study, the transgenic mice model has overcome these feared problems associated with the SIV model. Mice have the utmost advantages of being inexpensive, have high reproductive capacity and may be housed in large numbers in a fairly small facility [72]. Furthermore, conduct of experimentation can be done in large numbers and in replicates. The severe combined immune deficiency mouse engrafted with human peripheral blood mononuclear cells (hu-PBL-SCID) could be co-engrafted with xenografts containing the dual of human fetal thymus and liver tissue (SCID-hu thy/liv)/(SCID-hu thy/liv), and this model is widely applied in preclinical evaluation of antiretroviral therapy [55, 58]. In another study, Denton et al. supported these findings and showed that the female reproductive tissues in BLT mice are adequately reconstituted with

*3.3.3.2. BLT mice*

The human hematopoietic progenitor cells (CD34+) from human cord blood are used to reconstitute the immune system of immune-deficient mice also known as humanized mouse [41]. An additional feature or rather advancement incorporates a fragment of the fetal human thymus engraftment, which performs functionally as a human thymus. The significance of this is to allow for a more proper positive/negative T-cell selection previously not obtainable from the original model [74]. Immunologi and virologic parameters such as CD4+ cell depletion, extent of viremia, and co-receptor-mediated tropism were all observed in HIV infection of humanized experimental mice [74, 75]. The humice demonstrated transplanted human cells in mucosal linings therefore, most possibly get infected by intravaginal and/or intrarectal routes [76]. This model is used to evaluate novel approaches in HIV prevention and treatment options including human-neutralizing antibodies, usage of prophylactic anti-retroviral therapies, and T cell-specific siRNA transfer [77]. The effect of M. tuberculosis infection on the induction of HIV gene expression has been studied with HIV transgenic mice integrating the entire viral genome [78]. In this model, viral gene expression was triggered by M. tuberculosis and suppressed after anti-mycobacterial chemotherapy [78].
