Setting Up an Undergraduate Immunology Lab: Resources and Examples.

Laboratory courses in immunology require a different skill set for their development than lecture courses. They vary widely in their form based on factors like institutional budget and class size, and also in the prioritization of learning goals centered around reinforcing lecture concepts and/or building fundamental skills in the field of immunology. Lab activities can come from a variety of sources including published research protocols, commercial kits, computer-based tools or simulations, and case studies. Each has their own strengths, which will be explored here. There are also important decisions to make about how students will report their data, and what level of guidance in interpreting data is best to enhance student learning and growth. Finally, methods like use of rubrics can help ensure fair and efficient grading, especially with skills-based learning goals. Periodic assessment is important to ensure that activities contribute effectively to student learning and to guide improvements to the lab course over time.

Correction: Rapid Progressing Allele HLA-B35 Px Restricted Anti-HIV-1 CD8+ T Cells Recognize Vestigial CTL Epitopes.

[This corrects the article DOI: 10.1371/journal.pone.0010249.].

LINE-1 retrotransposable element DNA accumulates in HIV-1-infected cells.

Type 1 long-interspersed nuclear elements (L1s) are autonomous retrotransposable elements that retain the potential for activity in the human genome but are suppressed by host factors. Retrotransposition of L1s into chromosomal DNA can lead to genomic instability, whereas reverse transcription of L1 in the cytosol has the potential to activate innate immune sensors. We hypothesized that HIV-1 infection would compromise cellular control of L1 elements, resulting in the induction of retrotransposition events. Here, we show that HIV-1 infection enhances L1 retrotransposition in Jurkat cells in a Vif- and Vpr-dependent manner. In primary CD4(+) cells, HIV-1 infection results in the accumulation of L1 DNA, at least the majority of which is extrachromosomal. These data expose an unrecognized interaction between HIV-1 and endogenous retrotransposable elements, which may have implications for the innate immune response to HIV-1 infection, as well as for HIV-1-induced genomic instability and cytopathicity.

T cells target APOBEC3 proteins in human immunodeficiency virus type 1-infected humans and simian immunodeficiency virus-infected Indian rhesus macaques.

APOBEC3 proteins mediate potent antiretroviral activity by hypermutating the retroviral genome during reverse transcription. To counteract APOBEC3 and gain a replicative advantage, lentiviruses such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) have evolved the Vif protein, which targets APOBEC3 proteins for proteasomal degradation. However, the proteasome plays a critical role in the generation of T cell peptide epitopes. Whether Vif-mediated destruction of APOBEC3 proteins leads to the generation and presentation of APOBEC3-derived T cell epitopes on the surfaces of lentivirus-infected cells remains unknown. Here, using peptides derived from multiple Vif-sensitive APOBEC3 proteins, we identified APOBEC3-specific T cell responses in both HIV-1-infected patients and SIV-infected rhesus macaques. These results raise the possibility that these T cell responses may be part of the larger antiretroviral immune response.

HERV-K-specific T cells eliminate diverse HIV-1/2 and SIV primary isolates.

The genetic diversity of HIV-1 represents a major challenge in vaccine development. In this study, we establish a rationale for eliminating HIV-1-infected cells by targeting cellular immune responses against stable human endogenous retroviral (HERV) antigens. HERV DNA sequences in the human genome represent the remnants of ancient infectious retroviruses. We show that the infection of CD4+ T cells with HIV-1 resulted in transcription of the HML-2 lineage of HERV type K [HERV-K(HML-2)] and the expression of Gag and Env proteins. HERV-K(HML-2)-specific CD8+ T cells obtained from HIV-1-infected human subjects responded to HIV-1-infected cells in a Vif-dependent manner in vitro. Consistent with the proposed mode of action, a HERV-K(HML-2)-specific CD8+ T cell clone exhibited comprehensive elimination of cells infected with a panel of globally diverse HIV-1, HIV-2, and SIV isolates in vitro. We identified a second T cell response that exhibited cross-reactivity between homologous HIV-1-Pol and HERV-K(HML-2)-Pol determinants, raising the possibility that homology between HIV-1 and HERVs plays a role in shaping, and perhaps enhancing, the T cell response to HIV-1. This justifies the consideration of HERV-K(HML-2)-specific and cross-reactive T cell responses in the natural control of HIV-1 infection and for exploring HERV-K(HML-2)-targeted HIV-1 vaccines and immunotherapeutics.

Human endogenous retrovirus expression is inversely associated with chronic immune activation in HIV-1 infection.

Human endogenous retroviruses (HERV) are remnants of ancestral retroviral infections integrated into the germ line, and constitute approximately 8% of the genome. Several autoimmune disorders, malignancies, and infectious diseases such as HIV-1 are associated with higher HERV expression. The degree to which HERV expression in vivo results in persistent inflammation is not known. We studied the association of immune activation and HERV-K expression in 20 subjects with chronic, untreated progressive HIV-1 infection and 10 HIV-1 negative controls. The mean HERV-K gag and env RNA expression level in the HIV-1 infected cohort was higher than in the control group (p = 0.0003), and was negatively correlated with the frequency of activated CD38+HLA-DR+CD4+ T cells (Rho = -0.61; p = 0.01) and activated CD38+HLA-DR+CD8+ T cells (Rho = -0.51; p = 0.03). Although HIV-infected persons had higher levels of HERV-K RNA expression (as expected), the level of RNA expression was negatively associated with level of T cell activation. The mechanism for this unexpected association remains to be defined.

Influence of HAART on alternative reading frame immune responses over the course of HIV-1 infection.

BACKGROUND: Translational errors can result in bypassing of the main viral protein reading frames and the production of alternate reading frame (ARF) or cryptic peptides. Within HIV, there are many such ARFs in both sense and the antisense directions of transcription. These ARFs have the potential to generate immunogenic peptides called cryptic epitopes (CE). Both antiretroviral drug therapy and the immune system exert a mutational pressure on HIV-1. Immune pressure exerted by ARF CD8(+) T cells on the virus has already been observed in vitro. HAART has also been described to select HIV-1 variants for drug escape mutations. Since the mutational pressure exerted on one location of the HIV-1 genome can potentially affect the 3 reading frames, we hypothesized that ARF responses would be affected by this drug pressure in vivo. METHODOLOGY/PRINCIPAL FINDINGS: In this study we identified new ARFs derived from sense and antisense transcription of HIV-1. Many of these ARFs are detectable in circulating viral proteins. They are predominantly found in the HIV-1 env nucleotide region. We measured T cell responses to 199 HIV-1 CE encoded within 13 sense and 34 antisense HIV-1 ARFs. We were able to observe that these ARF responses are more frequent and of greater magnitude in chronically infected individuals compared to acutely infected patients, and in patients on HAART, the breadth of ARF responses increased. CONCLUSIONS/SIGNIFICANCE: These results have implications for vaccine design and unveil the existence of potential new epitopes that could be included as vaccine targets.

Human endogenous retrovirus K106 (HERV-K106) was infectious after the emergence of anatomically modern humans.

HERV-K113 and HERV-K115 have been considered to be among the youngest HERVs because they are the only known full-length proviruses that are insertionally polymorphic and maintain the open reading frames of their coding genes. However, recent data suggest that HERV-K113 is at least 800,000 years old, and HERV-K115 even older. A systematic study of HERV-K HML2 members to identify HERVs that may have infected the human genome in the more recent evolutionary past is lacking. Therefore, we sought to determine how recently HERVs were exogenous and infectious by examining sequence variation in the long terminal repeat (LTR) regions of all full-length HERV-K loci. We used the traditional method of inter-LTR comparison to analyze all full length HERV-Ks and determined that two insertions, HERV-K106 and HERV-K116 have no differences between their 5' and 3' LTR sequences, suggesting that these insertions were endogenized in the recent evolutionary past. Among these insertions with no sequence differences between their LTR regions, HERV-K106 had the most intact viral sequence structure. Coalescent analysis of HERV-K106 3' LTR sequences representing 51 ethnically diverse individuals suggests that HERV-K106 integrated into the human germ line approximately 150,000 years ago, after the emergence of anatomically modern humans.

Strong human endogenous retrovirus-specific T cell responses are associated with control of HIV-1 in chronic infection.

Eight percent of the human genome is composed of human endogenous retroviruses (HERVs), which are thought to be inactive remnants of ancient infections. Previously, we showed that individuals with early HIV-1 infection have stronger anti-HERV T cell responses than uninfected controls. In this study, we investigated whether these responses persist in chronic HIV-1 infection and whether they have a role in the control of HIV-1. Peripheral blood mononuclear cells (PBMCs) from 88 subjects diagnosed with HIV-1 infection for at least 1 year (median duration of diagnosis, 13 years) were tested for responses against HERV peptides in gamma interferon (IFN-γ) enzyme immunospot (ELISPOT) assays. Individuals who control HIV-1 viremia without highly active antiretroviral therapy (HAART) had stronger and broader HERV-specific T cell responses than HAART-suppressed patients, virologic noncontrollers, immunologic progressors, and uninfected controls (P < 0.05 for each pairwise comparison). In addition, the magnitude of the anti-HERV T cell response was inversely correlated with HIV-1 viral load (r(2) = 0.197, P = 0.0002) and associated with higher CD4(+) T cell counts (r(2) = 0.072, P = 0.027) in untreated patients. Flow cytometric analyses of an HLA-B51-restricted CD8(+) HERV response in one HIV-1-infected individual revealed a less activated and more differentiated phenotype than that stimulated by a homologous HIV-1 peptide. HLA-B51 tetramer dual staining within this individual confirmed two different T cell populations corresponding to these HERV and HIV-1 epitopes, ruling out cross-reactivity. These findings suggest a possible role for anti-HERV immunity in the control of chronic HIV-1 infection and provide support for a larger effort to design an HIV-1 vaccine that targets conserved antigens such as HERV.

Rapid progressing allele HLA-B35 Px restricted anti-HIV-1 CD8+ T cells recognize vestigial CTL epitopes.

BACKGROUND: The HLA-B*35-Px allele has been associated with rapid disease progression in HIV-1 infection, in contrast to the HLA-B*35-Py allele. METHODOLOGY/PRINCIPAL FINDINGS: Immune responses to two HLA-B*35 restricted HIV-1 specific CTL epitopes and their variants were followed longitudinally during early HIV-1 infection in 16 HLA-B*35+ individuals. Subjects expressing HLA-B*35-Px alleles showed no difference in response to the consensus epitopes compared to individuals with HLA-B*35-Py alleles. Surprisingly, all the HLA-B*35-Px+ individuals responded to epitope-variants even in the absence of a consensus response. Sequencing of the viral population revealed no evidence of variant virus in any of the individuals. CONCLUSIONS/SIGNIFICANCE: This demonstrates a novel phenomenon that distinguishes individuals with the HLA-B*35-Px rapid progressing allele and those with the HLA-B*35-Py slower progressing allele.

Cross-sectional dating of novel haplotypes of HERV-K 113 and HERV-K 115 indicate these proviruses originated in Africa before Homo sapiens.

The human genome, human endogenous retroviruses (HERV), of which HERV-K113 and HERV-K115 are the only known full-length proviruses that are insertionally polymorphic. Although a handful of previously published papers have documented their prevalence in the global population; to date, there has been no report on their prevalence in the United States population. Here, we studied the geographic distribution of K113 and K115 among 156 HIV-1+ subjects from the United States, including African Americans, Hispanics, and Caucasians. In the individuals studied, we found higher insertion frequencies of K113 (21%) and K115 (35%) in African Americans compared with Caucasians (K113 9% and K115 6%) within the United States. We also report the presence of three single nucleotide polymorphism sites in the K113 5' long terminal repeats (LTRs) and four in the K115 5' LTR that together constituted four haplotypes for K113 and five haplotypes for K115. HERV insertion times can be estimated from the sequence differences between the 5' and 3' LTR of each insertion, but this dating method cannot be used with HERV-K115. We developed a method to estimate insertion times by applying coalescent inference to 5' LTR sequences within our study population and validated this approach using an independent estimate derived from the genetic distance between K113 5' and 3' LTR sequences. Using our method, we estimated the insertion dates of K113 and K115 to be a minimum of 800,000 and 1.1 million years ago, respectively. Both these insertion dates predate the emergence of anatomically modern Homo sapiens.

Transcriptional errors in human immunodeficiency virus type 1 generate targets for T-cell responses.

We measured T-cell responses to human immunodeficiency virus type 1 (HIV-1) cryptic epitopes encoded by regions of the viral genome not normally translated into viral proteins. T-cell responses to cryptic epitopes and to regions normally spliced out of the HIV-1 viral proteins Rev and Tat were detected in HIV-1-infected subjects.

Characterization of ten novel Ty1/copia-like retrotransposon families of the grapevine genome.

BACKGROUND: Retrotransposons make a significant contribution to the size, organization and genetic diversity of their host genomes. To characterize retrotransposon families in the grapevine genome (the fourth crop plant genome sequenced) we have combined two approaches: a PCR-based method for the isolation of RnaseH-LTR sequences with a computer-based sequence similarity search in the whole-genome sequence of PN40024. RESULTS: Supported by a phylogenic analysis, ten novel Ty1/copia families were distinguished in this study. To select a canonical reference element sequence from amongst the various insertions in the genome belonging to each retroelement family, the following screening criteria were adopted to identify the element sequence with: (1) perfect 5 bp-duplication of target sites, (2) the highest level of identity between 5' and 3'-LTR within a single insertion sequence, and (3) longest, un-interrupted coding capacity within the gag-pol ORF. One to eight copies encoding a single putatively functional gag-pol polyprotein were found for three families, indicating that these families could be still autonomous and active. For the others, no autonomous copies were identified. However, a subset of copies within the presumably non-autonomous families had perfect identity between their 5' and 3' LTRs, indicating a recent insertion event. A phylogenic study based on the sequence alignment of the region located between reverse transcriptase domains I and VII distinguished these 10 families from other plant retrotransposons. Including the previously characterized Ty1/copia-like grapevine retrotransposons Tvv1 and Vine 1 and the Ty3/gypsy-like Gret1 in this assessment, a total of 1709 copies were identified for the 13 retrotransposon families, representing 1.24% of the sequenced genome. The copy number per family ranged from 91-212 copies. We performed insertion site profiling for 8 out of the 13 retrotransposon families and confirmed multiple insertions of these elements across the Vitis genus. Insertional polymorphism analysis and dating of full-length copies based on their LTR divergence demonstrated that each family has a particular amplification history, with 71% of the identified copies being inserted within the last 2 million years. CONCLUSION: The strategy we used efficiently delivered new Ty1/copia-like retrotransposon sequences, increasing the total number of characterized grapevine retrotrotransposons from 3 to 13. We provide insights into the representation and dynamics of the 13 families in the genome. Our data demonstrated that each family has a particular amplification pattern, with 7 families having copies recently inserted within the last 0.2 million year. Among those 7 families with recent insertions, three retain the capacity for activity in the grape genome today.

Nucleoside analogue reverse transcriptase inhibitors differentially inhibit human LINE-1 retrotransposition.

BACKGROUND: Intact LINE-1 elements are the only retrotransposons encoded by the human genome known to be capable of autonomous replication. Numerous cases of genetic disease have been traced to gene disruptions caused by LINE-1 retrotransposition events in germ-line cells. In addition, genomic instability resulting from LINE-1 retrotransposition in somatic cells has been proposed as a contributing factor to oncogenesis and to cancer progression. LINE-1 element activity may also play a role in normal physiology. METHODS AND PRINCIPAL FINDINGS: Using an in vitro LINE-1 retrotransposition reporter assay, we evaluated the abilities of several antiretroviral compounds to inhibit LINE-1 retrotransposition. The nucleoside analogue reverse transcriptase inhibitors (nRTIs): stavudine, zidovudine, tenofovir disoproxil fumarate, and lamivudine all inhibited LINE-1 retrotransposition with varying degrees of potencies, while the non-nucleoside HIV-1 reverse transcriptase inhibitor nevirapine showed no effect. CONCLUSIONS/SIGNIFICANCE: Our data demonstrates the ability for nRTIs to suppress LINE-1 retrotransposition. This is immediately applicable to studies aimed at examining potential roles for LINE-1 retrotransposition in physiological processes. In addition, our data raises novel safety considerations for nRTIs based on their potential to disrupt physiological processes involving LINE-1 retrotransposition.

T cell responses to human endogenous retroviruses in HIV-1 infection.

Human endogenous retroviruses (HERVs) are remnants of ancient infectious agents that have integrated into the human genome. Under normal circumstances, HERVs are functionally defective or controlled by host factors. In HIV-1-infected individuals, intracellular defense mechanisms are compromised. We hypothesized that HIV-1 infection would remove or alter controls on HERV activity. Expression of HERV could potentially stimulate a T cell response to HERV antigens, and in regions of HIV-1/HERV similarity, these T cells could be cross-reactive. We determined that the levels of HERV production in HIV-1-positive individuals exceed those of HIV-1-negative controls. To investigate the impact of HERV activity on specific immunity, we examined T cell responses to HERV peptides in 29 HIV-1-positive and 13 HIV-1-negative study participants. We report T cell responses to peptides derived from regions of HERV detected by ELISPOT analysis in the HIV-1-positive study participants. We show an inverse correlation between anti-HERV T cell responses and HIV-1 plasma viral load. In HIV-1-positive individuals, we demonstrate that HERV-specific T cells are capable of killing cells presenting their cognate peptide. These data indicate that HIV-1 infection leads to HERV expression and stimulation of a HERV-specific CD8+ T cell response. HERV-specific CD8+ T cells have characteristics consistent with an important role in the response to HIV-1 infection: a phenotype similar to that of T cells responding to an effectively controlled virus (cytomegalovirus), an inverse correlation with HIV-1 plasma viral load, and the ability to lyse cells presenting their target peptide. These characteristics suggest that elicitation of anti-HERV-specific immune responses is a novel approach to immunotherapeutic vaccination. As endogenous retroviral sequences are fixed in the human genome, they provide a stable target, and HERV-specific T cells could recognize a cell infected by any HIV-1 viral variant. HERV-specific immunity is an important new avenue for investigation in HIV-1 pathogenesis and vaccine design.

HIV-1/HSV-2 co-infected adults in early HIV-1 infection have elevated CD4+ T cell counts.

INTRODUCTION: HIV-1 is often acquired in the presence of pre-existing co-infections, such as Herpes Simplex Virus 2 (HSV-2). We examined the impact of HSV-2 status at the time of HIV-1 acquisition for its impact on subsequent clinical course, and total CD4+ T cell phenotypes. METHODS: We assessed the relationship of HSV-1/HSV-2 co-infection status on CD4+ T cell counts and HIV-1 RNA levels over time prior in a cohort of 186 treatment naïve adults identified during early HIV-1 infection. We assessed the activation and differentiation state of total CD4+ T cells at study entry by HSV-2 status. RESULTS: Of 186 recently HIV-1 infected persons, 101 (54%) were sero-positive for HSV-2. There was no difference in initial CD8+ T cell count, or differences between the groups for age, gender, or race based on HSV-2 status. Persons with HIV-1/HSV-2 co-infection sustained higher CD4+ T cell counts over time (+69 cells/ul greater (SD = 33.7, p = 0.04) than those with HIV-1 infection alone (Figure 1), after adjustment for HIV-1 RNA levels (-57 cells per 1 log(10) higher HIV-1 RNA, p<0.0001). We did not observe a relationship between HSV-2 infection status with plasma HIV-1 RNA levels over time. HSV-2 acquisition after HIV-1 acquisition had no impact on CD4+ count or viral load. We did not detect differences in CD4+ T cell activation or differentiation state by HSV-2+ status. DISCUSSION: We observed no effect of HSV-2 status on viral load. However, we did observe that treatment naïve, recently HIV-1 infected adults co-infected with HSV-2+ at the time of HIV-1 acquisition had higher CD4+ T cell counts over time. If verified in other cohorts, this result poses a striking paradox, and its public health implications are not immediately clear.